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Sample records for glucose phosphate isomerase

  1. Glucose-6-phosphate isomerase.

    PubMed

    Achari, A; Marshall, S E; Muirhead, H; Palmieri, R H; Noltmann, E A

    1981-06-26

    Glucose-6-phosphate isomerase (EC 5.3.1.9) is a dimeric enzyme of molecular mass 132000 which catalyses the interconversion of D-glucose-6-phosphate and D-fructose-6-phosphate. The crystal structure of the enzyme from pig muscle has been determined at a nominal resolution of 2.6 A. The structure is of the alpha/beta type. Each subunit consists of two domains and the active site is in both the domain interface and the subunit interface (P.J. Shaw & H. Muirhead (1976), FEBS Lett. 65, 50-55). Each subunit contains 13 methionine residues so that cyanogen bromide cleavage will produce 14 fragments, most of which have been identified and at least partly purified. Sequence information is given for about one-third of the molecule from 5 cyanogen bromide fragments. One of the sequences includes a modified lysine residue. Modification of this residue leads to a parallel loss of enzymatic activity. A tentative fit of two of the peptides to the electron density map has been made. It seems possible that glucose-6-phosphate isomerase, triose phosphate isomerase and pyruvate kinase all contain a histidine and a glutamate residue at the active site. PMID:6115414

  2. Genetics Home Reference: glucose phosphate isomerase deficiency

    MedlinePlus

    ... Me Understand Genetics Home Health Conditions GPI deficiency glucose phosphate isomerase deficiency Enable Javascript to view the ... boxes. Download PDF Open All Close All Description Glucose phosphate isomerase (GPI) deficiency is an inherited disorder ...

  3. Human glucose phosphate isomerase: Exon mapping and gene structure

    SciTech Connect

    Xu, Weiming; Lee, Pauline; Beutler, E.

    1995-10-10

    The structure of the gene for human glucose phosphate isomerase (GPI) has been determined. Three GPI clones were isolated from a human genomic library by using a full-length GPI cDNA probe and were characterized. Oligonucleotides based on the known cDNA sequence were used as primers in amplification and sequence analyses. This led to the identification of the exon-intron junctions. By this approach, 18 exons and 17 introns have been identified. The exons range in size from 44 to 431 nucleotides. The intronic sequences surrounding the exons provide useful information for the identification of mutations that give rise to human GPI deficiency associated with chronic hemolytic anemia. 13 refs., 4 figs., 1 tab.

  4. 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. PMID:27014866

  5. Survival of glucose phosphate isomerase null somatic cells and germ cells in adult mouse chimaeras.

    PubMed

    Keighren, Margaret A; Flockhart, Jean H; West, John D

    2016-01-01

    The mouse Gpi1 gene encodes the glycolytic enzyme glucose phosphate isomerase. Homozygous Gpi1(-/-) null mouse embryos die but a previous study showed that some homozygous Gpi1(-/-) null cells survived when combined with wild-type cells in fetal chimaeras. One adult female Gpi1(-/-)↔Gpi1(c/c) chimaera with functional Gpi1(-/-) null oocytes was also identified in a preliminary study. The aims were to characterise the survival of Gpi1(-/-) null cells in adult Gpi1(-/-)↔Gpi1(c/c) chimaeras and determine if Gpi1(-/-) null germ cells are functional. Analysis of adult Gpi1(-/-)↔Gpi1(c/c) chimaeras with pigment and a reiterated transgenic lineage marker showed that low numbers of homozygous Gpi1(-/-) null cells could survive in many tissues of adult chimaeras, including oocytes. Breeding experiments confirmed that Gpi1(-/-) null oocytes in one female Gpi1(-/-)↔Gpi1(c/c) chimaera were functional and provided preliminary evidence that one male putative Gpi1(-/-)↔Gpi1(c/c) chimaera produced functional spermatozoa from homozygous Gpi1(-/-) null germ cells. Although the male chimaera was almost certainly Gpi1(-/-)↔Gpi1(c/c), this part of the study is considered preliminary because only blood was typed for GPI. Gpi1(-/-) null germ cells should survive in a chimaeric testis if they are supported by wild-type Sertoli cells. It is also feasible that spermatozoa could bypass a block at GPI, but not blocks at some later steps in glycolysis, by using fructose, rather than glucose, as the substrate for glycolysis. Although chimaera analysis proved inefficient for studying the fate of Gpi1(-/-) null germ cells, it successfully identified functional Gpi1(-/-) null oocytes and revealed that some Gpi1(-/-) null cells could survive in many adult tissues. PMID:27103217

  6. Survival of glucose phosphate isomerase null somatic cells and germ cells in adult mouse chimaeras

    PubMed Central

    Keighren, Margaret A.; Flockhart, Jean H.

    2016-01-01

    ABSTRACT The mouse Gpi1 gene encodes the glycolytic enzyme glucose phosphate isomerase. Homozygous Gpi1−/− null mouse embryos die but a previous study showed that some homozygous Gpi1−/− null cells survived when combined with wild-type cells in fetal chimaeras. One adult female Gpi1−/−↔Gpi1c/c chimaera with functional Gpi1−/− null oocytes was also identified in a preliminary study. The aims were to characterise the survival of Gpi1−/− null cells in adult Gpi1−/−↔Gpi1c/c chimaeras and determine if Gpi1−/− null germ cells are functional. Analysis of adult Gpi1−/−↔Gpi1c/c chimaeras with pigment and a reiterated transgenic lineage marker showed that low numbers of homozygous Gpi1−/− null cells could survive in many tissues of adult chimaeras, including oocytes. Breeding experiments confirmed that Gpi1−/− null oocytes in one female Gpi1−/−↔Gpi1c/c chimaera were functional and provided preliminary evidence that one male putative Gpi1−/−↔Gpi1c/c chimaera produced functional spermatozoa from homozygous Gpi1−/− null germ cells. Although the male chimaera was almost certainly Gpi1−/−↔Gpi1c/c, this part of the study is considered preliminary because only blood was typed for GPI. Gpi1−/− null germ cells should survive in a chimaeric testis if they are supported by wild-type Sertoli cells. It is also feasible that spermatozoa could bypass a block at GPI, but not blocks at some later steps in glycolysis, by using fructose, rather than glucose, as the substrate for glycolysis. Although chimaera analysis proved inefficient for studying the fate of Gpi1−/− null germ cells, it successfully identified functional Gpi1−/− null oocytes and revealed that some Gpi1−/− null cells could survive in many adult tissues. PMID:27103217

  7. Identification of a novel tandemly repeated sequence present in an intron of the glucose phosphate isomerase (GPI) gene in mouse and man

    SciTech Connect

    Faik, P.; Walker, J.I.H.; Morgan, M.J. )

    1994-05-01

    Glucose phosphate isomerase (GPI, glucose 6-phosphate ketol-isomerase, EC 5.3.1.9) is a housekeeping gene expressed in all tissues and organisms that utilize glycolysis and gluconeogenesis. Deficiency in humans leads to a rare form of nonspherocytic hemolytic anemia. The authors have isolated a 3.2-kb mouse cDNA containing glucose phosphate isomerase coding sequence and a 2.1-kb intronic sequence and a large proportion of the human gene (approaching 55 kb) in four phage [lambda] recombinants. A 4-kb intronic fragment from the human gene showing homology to the mouse intronic sequence has been isolated and sequenced. The fragment contains approximately 1.5 kb of sequence that is composited of 30 repeat units of a novel 50-kb tandemly repeated unit. The mouse intronic sequence contains 18 similar units. The human consensus sequence differs from the mouse consensus sequence at only 7 positions out of 50 (positions 16, 26, 27, 42, 43, 47, and 48). A probe containing the repeat element detects polymorphisms, specific to glucose phosphate isomerase, in human DNA. The repeat element does not appear to be present at any other loci in human DNA. The conservation of this intronic repeat element extends to pig and Chinese hamster. 26 refs., 4 figs.

  8. 21 CFR 862.1720 - Triose phosphate isomerase test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... isomerase test system is a device intended to measure the activity of the enzyme triose phosphate isomerase in erythrocytes (red blood cells). Triose phosphate isomerase is an enzyme important in glycolysis... this device are used in the diagnosis and treatment of congenital triose phosphate isomerase...

  9. 21 CFR 862.1720 - Triose phosphate isomerase test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... isomerase test system is a device intended to measure the activity of the enzyme triose phosphate isomerase in erythrocytes (red blood cells). Triose phosphate isomerase is an enzyme important in glycolysis... this device are used in the diagnosis and treatment of congenital triose phosphate isomerase...

  10. 21 CFR 862.1720 - Triose phosphate isomerase test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... isomerase test system is a device intended to measure the activity of the enzyme triose phosphate isomerase in erythrocytes (red blood cells). Triose phosphate isomerase is an enzyme important in glycolysis... this device are used in the diagnosis and treatment of congenital triose phosphate isomerase...

  11. 21 CFR 862.1720 - Triose phosphate isomerase test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... isomerase test system is a device intended to measure the activity of the enzyme triose phosphate isomerase in erythrocytes (red blood cells). Triose phosphate isomerase is an enzyme important in glycolysis... this device are used in the diagnosis and treatment of congenital triose phosphate isomerase...

  12. 21 CFR 862.1720 - Triose phosphate isomerase test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... isomerase test system is a device intended to measure the activity of the enzyme triose phosphate isomerase in erythrocytes (red blood cells). Triose phosphate isomerase is an enzyme important in glycolysis... this device are used in the diagnosis and treatment of congenital triose phosphate isomerase...

  13. Thermoinactivation Mechanism of Glucose Isomerase

    NASA Astrophysics Data System (ADS)

    Lim, Leng Hong; Saville, Bradley A.

    In this article, the mechanisms of thermoinactivation of glucose isomerase (GI) from Streptomyces rubiginosus (in soluble and immobilized forms) were investigated, particularly the contributions of thiol oxidation of the enzyme's cysteine residue and a "Maillard-like" reaction between the enzyme and sugars in high fructose corn syrup (HFCS). Soluble GI (SGI) was successfully immobilized on silica gel (13.5 μm particle size), with an activity yield between 20 and 40%. The immobilized GI (IGI) has high enzyme retention on the support during the glucose isomerization process. In batch reactors, SGI (half-life =145 h) was more stable than IGI (half-life=27 h) at 60°C in HFCS, whereas at 80°C, IGI (half-life=12 h) was more stable than SGI (half-life=5.2 h). IGI was subject to thiol oxidation at 60°C, which contributed to the enzyme's deactivation. IGI was subject to thiol oxidation at 80°C, but this did not contribute to the deactivation of the enzyme. SGI did not undergo thiol oxidation at 60°C, but at 80°C SGI underwent severe precipitation and thiol oxidation, which caused the enzyme to deactivate. Experimental results show that immobilization suppresses the destablizing effect of thiol oxidation on GI. A "Maillard-like" reaction between SGI and the sugars also caused SGI thermoinactivation at 60, 70, and 80°C, but had minimal effect on IGI. At 60 and 80°C, IGI had higher thermostability in continuous reactors than in batch reactors, possibily because of reduced contact with deleterious compounds in HFCS.

  14. 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

  15. 21 CFR 184.1372 - Insoluble glucose isomerase enzyme preparations.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Insoluble glucose isomerase enzyme preparations... Substances Affirmed as GRAS § 184.1372 Insoluble glucose isomerase enzyme preparations. (a) Insoluble glucose isomerase enzyme preparations are used in the production of high fructose corn syrup described in §...

  16. 21 CFR 184.1372 - Insoluble glucose isomerase enzyme preparations.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Insoluble glucose isomerase enzyme preparations... RECOGNIZED AS SAFE Listing of Specific Substances Affirmed as GRAS § 184.1372 Insoluble glucose isomerase enzyme preparations. (a) Insoluble glucose isomerase enzyme preparations are used in the production...

  17. 21 CFR 184.1372 - Insoluble glucose isomerase enzyme preparations.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Insoluble glucose isomerase enzyme preparations... RECOGNIZED AS SAFE Listing of Specific Substances Affirmed as GRAS § 184.1372 Insoluble glucose isomerase enzyme preparations. (a) Insoluble glucose isomerase enzyme preparations are used in the production...

  18. 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 ...

  19. 21 CFR 184.1372 - Insoluble glucose isomerase enzyme preparations.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Insoluble glucose isomerase enzyme preparations... enzyme preparations. (a) Insoluble glucose isomerase enzyme preparations are used in the production of... additional requirements for enzyme preparations in the Food Chemicals Codex, 3d Ed. (1981), p. 107, which...

  20. 21 CFR 184.1372 - Insoluble glucose isomerase enzyme preparations.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Insoluble glucose isomerase enzyme preparations... enzyme preparations. (a) Insoluble glucose isomerase enzyme preparations are used in the production of... additional requirements for enzyme preparations in the Food Chemicals Codex, 3d Ed. (1981), p. 107, which...

  1. Molecular and industrial aspects of glucose isomerase.

    PubMed Central

    Bhosale, S H; Rao, M B; Deshpande, V V

    1996-01-01

    Glucose isomerase (GI) (D-xylose ketol-isomerase; EC. 5.3.1.5) catalyzes the reversible isomerization of D-glucose and D-xylose to D-fructose and D-xylulose, respectively. The enzyme has the largest market in the food industry because of its application in the production of high-fructose corn syrup (HFCS). HFCS, an equilibrium mixture of glucose and fructose, is 1.3 times sweeter than sucrose and serves as a sweetener for use by diabetics. Interconversion of xylose to xylulose by GI serves a nutritional requirement in saprophytic bacteria and has a potential application in the bioconversion of hemicellulose to ethanol. The enzyme is widely distributed in prokaryotes. Intensive research efforts are directed toward improving its suitability for industrial application. Development of microbial strains capable of utilizing xylan-containing raw materials for growth or screening for constitutive mutants of GI is expected to lead to discontinuation of the use of xylose as an inducer for the production of the enzyme. Elimination of Co2+ from the fermentation medium is desirable for avoiding health problems arising from human consumption of HFCS. Immobilization of GI provides an efficient means for its easy recovery and reuse and lowers the cost of its use. X-ray crystallographic and genetic engineering studies support a hydride shift mechanism for the action of GI. Cloning of GI in homologous as well as heterologous hosts has been carried out, with the prime aim of overproducing the enzyme and deciphering the genetic organization of individual genes (xylA, xylB, and xylR) in the xyl operon of different microorganisms. The organization of xylA and xylB seems to be highly conserved in all bacteria. The two genes are transcribed from the same strand in Escherichia coli and Bacillus and Lactobacillus species, whereas they are transcribed divergently on different strands in Streptomyces species. A comparison of the xylA sequences from several bacterial sources revealed the

  2. Ribose 5-Phosphate Isomerase Investigations for the Undergraduate Biochemistry Laboratory

    ERIC Educational Resources Information Center

    Jewett, Kathy; Sandwick, Roger K.

    2011-01-01

    The enzyme ribose 5-phosphate isomerase (RpiA) has many features that make it attractive as a focal point of a semester-long, advanced biochemistry laboratory for undergraduate students. The protein can easily and inexpensively be isolated from spinach using traditional purification techniques. Characterization of RpiA enzyme activity can be…

  3. The amino acid sequence of rabbit muscle triose phosphate isomerase.

    PubMed Central

    Corran, P H; Waley, S G

    1975-01-01

    The amino acid sequence of rabbit muscle triose phosphate isomerase was deduced by characterizing peptides that overlap the tryptic peptides. Thiol groups were modified by oxidation, carboxymethylation or aminoen. About 50 peptides that provided information about overlaps were isolated; the peptides were mostly characterized by their compositions and N-terminal residues. The peptide chains contain 248 amino acid residues, and no evidence for dissimilarity of the two subunits that comprise the native enzyme was found. The sequence of the rabbit muscle enzyme may be compared with that of the coelacanth enzyme (Kolb et al., 1974): 84% of the residues are in identical positions. Similarly, comparison of the sequence with that inferred for the chicken enzyme (Furth et al., 1974) shows that 87% of the residues are in identical positions. Limited though these comparisons are, they suggest that triose phosphate isomerase has one of the lowest rates of evolutionary change. An extended version of the present paper has been deposited as Supplementary Publication SUP 50040 (42 pages) at the British Library (Lending Division) (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms given in Biochem. J. (1975) 145, 5. PMID:1171682

  4. Characterization of a mutant glucose isomerase from Thermoanaerobacterium saccharolyticum.

    PubMed

    Xu, Heng; Shen, Dong; Wu, Xue-Qiang; Liu, Zhi-Wei; Yang, Qi-He

    2014-10-01

    A series of site-directed mutant glucose isomerase at tryptophan 139 from Thermoanaerobacterium saccharolyticum strain B6A were purified to gel electrophoretic homogeneity, and the biochemical properties were determined. W139F mutation is the most efficient mutant derivative with a tenfold increase in its catalytic efficiency toward glucose compared with the native GI. With a maximal activity at 80 °C of 59.58 U/mg on glucose, this mutant derivative is the most active type ever reported. The enzyme activity was maximal at 90 °C and like other glucose isomerase, this mutant enzyme required Co(2+) or Mg(2+) for enzyme activity and thermal stability (stable for 20 h at 80 °C in the absence of substrate). Its optimum pH was around 7.0, and it had 86 % of its maximum activity at pH 6.0 incubated for 12 h at 60 °C. This enzyme was determined as thermostable and weak-acid stable. These findings indicated that the mutant GI W139F from T. saccharolyticum strain B6A is appropriate for use as a potential candidate for high-fructose corn syrup producing enzyme. PMID:25139657

  5. Ribose 5-Phosphate Isomerase B Knockdown Compromises Trypanosoma brucei Bloodstream Form Infectivity

    PubMed Central

    Loureiro, Inês; Faria, Joana; Clayton, Christine; Macedo-Ribeiro, Sandra; Santarém, Nuno; Roy, Nilanjan; Cordeiro-da-Siva, Anabela; Tavares, Joana

    2015-01-01

    Ribose 5-phosphate isomerase is an enzyme involved in the non-oxidative branch of the pentose phosphate pathway, and catalyzes the inter-conversion of D-ribose 5-phosphate and D-ribulose 5-phosphate. Trypanosomatids, including the agent of African sleeping sickness namely Trypanosoma brucei, have a type B ribose-5-phosphate isomerase. This enzyme is absent from humans, which have a structurally unrelated ribose 5-phosphate isomerase type A, and therefore has been proposed as an attractive drug target waiting further characterization. In this study, Trypanosoma brucei ribose 5-phosphate isomerase B showed in vitro isomerase activity. RNAi against this enzyme reduced parasites' in vitro growth, and more importantly, bloodstream forms infectivity. Mice infected with induced RNAi clones exhibited lower parasitaemia and a prolonged survival compared to control mice. Phenotypic reversion was achieved by complementing induced RNAi clones with an ectopic copy of Trypanosoma cruzi gene. Our results present the first functional characterization of Trypanosoma brucei ribose 5-phosphate isomerase B, and show the relevance of an enzyme belonging to the non-oxidative branch of the pentose phosphate pathway in the context of Trypanosoma brucei infection. PMID:25568941

  6. The tryptic peptides of rabbit muscle triose phosphate isomerase

    PubMed Central

    Corran, P. H.; Waley, S. G.

    1974-01-01

    1. The peptides obtained by tryptic digestion of S-[14C]carboxymethylated rabbit muscle triose phosphate isomerase have been studied. 2. The first step in the fractionation of the tryptic digest was gel filtration on coupled columns of Sephadex G-25 and G-50. Further fractionation was carried out by paper electrophoresis and paper chromatography. 3. The digest contained 26 peptides and three free amino acids. The sizes of the peptides ranged from two to 29 residues. 4. The sequences of the peptides have been determined. 5. The length of the polypeptide chains is about 250 amino acid residues. 6. The variant sequences encountered were due to partial deamidation; this may be one of the reasons for multiple forms of the enzyme. 7. The chicken and rabbit enzymes are compared. 8. Detailed evidence for the sequences of the tryptic peptides has been deposited as Supplementary Publication SUP 50024 at the British Library, Lending Division (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms given in Biochem. J. (1973) 131, 5. PMID:4618774

  7. Development of Novel Sugar Isomerases by Optimization of Active Sites in Phosphosugar Isomerases for Monosaccharides

    PubMed Central

    Yeom, Soo-Jin; Kim, Yeong-Su

    2013-01-01

    Phosphosugar isomerases can catalyze the isomerization of not only phosphosugar but also of monosaccharides, suggesting that the phosphosugar isomerases can be used as sugar isomerases that do not exist in nature. Determination of active-site residues of phosphosugar isomerases, including ribose-5-phosphate isomerase from Clostridium difficile (CDRPI), mannose-6-phosphate isomerase from Bacillus subtilis (BSMPI), and glucose-6-phosphate isomerase from Pyrococcus furiosus (PFGPI), was accomplished by docking of monosaccharides onto the structure models of the isomerases. The determinant residues, including Arg133 of CDRPI, Arg192 of BSMPI, and Thr85 of PFGPI, were subjected to alanine substitutions and found to act as phosphate-binding sites. R133D of CDRPI, R192 of BSMPI, and T85Q of PFGPI displayed the highest catalytic efficiencies for monosaccharides at each position. These residues exhibited 1.8-, 3.5-, and 4.9-fold higher catalytic efficiencies, respectively, for the monosaccharides than the wild-type enzyme. However, the activities of these 3 variant enzymes for phosphosugars as the original substrates disappeared. Thus, R133D of CDRPI, R192 of BSMPI, and T85Q of PFGPI are no longer phosphosugar isomerases; instead, they are changed to a d-ribose isomerase, an l-ribose isomerase, and an l-talose isomerase, respectively. In this study, we used substrate-tailored optimization to develop novel sugar isomerases which are not found in nature based on phosphosugar isomerases. PMID:23204422

  8. Amino acid sequences around the cysteine residues of rabbit muscle triose phosphate isomerase

    PubMed Central

    Miller, Janet C.; Waley, S. G.

    1971-01-01

    1. The nature of the subunits in rabbit muscle triose phosphate isomerase has been investigated. 2. Amino acid analyses show that there are five cysteine residues and two methionine residues/subunit. 3. The amino acid sequences around the cysteine residues have been determined; these account for about 75 residues. 4. Cleavage at the methionine residues with cyanogen bromide gave three fragments. 5. These results show that the subunits correspond to polypeptide chains, containing about 230 amino acid residues. The chains in triose phosphate isomerase seem to be shorter than those of other glycolytic enzymes. PMID:5165707

  9. Erythritol feeds the pentose phosphate pathway via three new isomerases leading to D-erythrose-4-phosphate in Brucella

    PubMed Central

    Barbier, Thibault; Collard, François; Zúñiga-Ripa, Amaia; Moriyón, Ignacio; Godard, Thibault; Becker, Judith; Wittmann, Christoph; Van Schaftingen, Emile; Letesson, Jean-Jacques

    2014-01-01

    Erythritol is an important nutrient for several α-2 Proteobacteria, including N2-fixing plant endosymbionts and Brucella, a worldwide pathogen that finds this four-carbon polyol in genital tissues. Erythritol metabolism involves phosphorylation to l-erythritol-4-phosphate by the kinase EryA and oxidation of the latter to l-3-tetrulose 4-phosphate by the dehydrogenase EryB. It is accepted that further steps involve oxidation by the putative dehydrogenase EryC and subsequent decarboxylation to yield triose-phosphates. Accordingly, growth on erythritol as the sole C source should require aldolase and fructose-1,6-bisphosphatase to produce essential hexose-6-monophosphate. However, we observed that a mutant devoid of fructose-1,6-bisphosphatases grew normally on erythritol and that EryC, which was assumed to be a dehydrogenase, actually belongs to the xylose isomerase superfamily. Moreover, we found that TpiA2 and RpiB, distant homologs of triose phosphate isomerase and ribose 5-phosphate isomerase B, were necessary, as previously shown for Rhizobium. By using purified recombinant enzymes, we demonstrated that l-3-tetrulose-4-phosphate was converted to d-erythrose 4-phosphate through three previously unknown isomerization reactions catalyzed by EryC (tetrulose-4-phosphate racemase), TpiA2 (d-3-tetrulose-4-phosphate isomerase; renamed EryH), and RpiB (d-erythrose-4-phosphate isomerase; renamed EryI), a pathway fully consistent with the isotopomer distribution of the erythrose-4-phosphate-derived amino acids phenylalanine and tyrosine obtained from bacteria grown on 13C-labeled erythritol. d-Erythrose-4-phosphate is then converted by enzymes of the pentose phosphate pathway to glyceraldehyde 3-phosphate and fructose 6-phosphate, thus bypassing fructose-1,6-bisphosphatase. This is the first description to our knowledge of a route feeding carbohydrate metabolism exclusively via d-erythrose 4-phosphate, a pathway that may provide clues to the preferential metabolism of

  10. Erythritol feeds the pentose phosphate pathway via three new isomerases leading to D-erythrose-4-phosphate in Brucella.

    PubMed

    Barbier, Thibault; Collard, François; Zúñiga-Ripa, Amaia; Moriyón, Ignacio; Godard, Thibault; Becker, Judith; Wittmann, Christoph; Van Schaftingen, Emile; Letesson, Jean-Jacques

    2014-12-16

    Erythritol is an important nutrient for several α-2 Proteobacteria, including N2-fixing plant endosymbionts and Brucella, a worldwide pathogen that finds this four-carbon polyol in genital tissues. Erythritol metabolism involves phosphorylation to L-erythritol-4-phosphate by the kinase EryA and oxidation of the latter to L-3-tetrulose 4-phosphate by the dehydrogenase EryB. It is accepted that further steps involve oxidation by the putative dehydrogenase EryC and subsequent decarboxylation to yield triose-phosphates. Accordingly, growth on erythritol as the sole C source should require aldolase and fructose-1,6-bisphosphatase to produce essential hexose-6-monophosphate. However, we observed that a mutant devoid of fructose-1,6-bisphosphatases grew normally on erythritol and that EryC, which was assumed to be a dehydrogenase, actually belongs to the xylose isomerase superfamily. Moreover, we found that TpiA2 and RpiB, distant homologs of triose phosphate isomerase and ribose 5-phosphate isomerase B, were necessary, as previously shown for Rhizobium. By using purified recombinant enzymes, we demonstrated that L-3-tetrulose-4-phosphate was converted to D-erythrose 4-phosphate through three previously unknown isomerization reactions catalyzed by EryC (tetrulose-4-phosphate racemase), TpiA2 (D-3-tetrulose-4-phosphate isomerase; renamed EryH), and RpiB (D-erythrose-4-phosphate isomerase; renamed EryI), a pathway fully consistent with the isotopomer distribution of the erythrose-4-phosphate-derived amino acids phenylalanine and tyrosine obtained from bacteria grown on (13)C-labeled erythritol. D-erythrose-4-phosphate is then converted by enzymes of the pentose phosphate pathway to glyceraldehyde 3-phosphate and fructose 6-phosphate, thus bypassing fructose-1,6-bisphosphatase. This is the first description to our knowledge of a route feeding carbohydrate metabolism exclusively via D-erythrose 4-phosphate, a pathway that may provide clues to the preferential metabolism of

  11. Purification and characterization of thermostable glucose isomerase from Clostridium thermosulfurogenes and Thermoanaerobacter strain B6A.

    PubMed Central

    Lee, C Y; Zeikus, J G

    1991-01-01

    Glucose isomerases produced by Thermoanaerobacter strain B6A and Clostridium thermosulfurogenes strain 4B were purified 10-11-fold to homogeneity and their physicochemical and catalytic properties were determined. Both purified enzymes displayed very similar properties (native Mr 200,000, tetrameric subunit composition, and apparent pH optima 7.0-7.5). The enzymes were stable at pH 5.5-12.0, and maintained more than 90% activity after incubation at high temperature (85 degrees C) for 1 h in the presence of metal ions. The N-terminal amino acid sequences of both thermostable glucose isomerases were Met-Asn-Lys-Tyr-Phe-Glu-Asn and were not similar to that of the thermolabile Bacillus subtilis enzyme. The glucose isomerase from C. thermosulfurogenes and Thermoanaerobacter displayed pI values of 4.9 and 4.8, and their kcat. and Km values for D-glucose at 65 degrees C were 1040 and 1260 min-1 and 140 and 120 mM respectively. Both enzymes displayed higher kcat. and lower Km values for D-xylose than for D-glucose. The C. thermosulfurogenes enzyme required Co2+ or Mg2+ for thermal stability and glucose isomerase activity, and Mn2+ or these metals for xylose isomerase activity. Crystals of C. thermosulfurogenes glucose isomerase were formed at room temperature by the hanging-drop method using 16-18% poly(ethylene glycol) (PEG) 4000 in 0.1 M-citrate buffer. Images Fig. 1. Fig. 5. PMID:1996956

  12. Glucose-6-Phosphate Dehydrogenase Deficiency.

    PubMed

    Luzzatto, Lucio; Nannelli, Caterina; Notaro, Rosario

    2016-04-01

    G6PD is a housekeeping gene expressed in all cells. Glucose-6-phosphate dehydrogenase (G6PD) is part of the pentose phosphate pathway, and its main physiologic role is to provide NADPH. G6PD deficiency, one of the commonest inherited enzyme abnormalities in humans, arises through one of many possible mutations, most of which reduce the stability of the enzyme and its level as red cells age. G6PD-deficient persons are mostly asymptomatic, but they can develop severe jaundice during the neonatal period and acute hemolytic anemia when they ingest fava beans or when they are exposed to certain infections or drugs. G6PD deficiency is a global health issue. PMID:27040960

  13. Analysis of the Arabinose-5-Phosphate Isomerase of Bacteroides fragilis Provides Insight into Regulation of Single-Domain Arabinose Phosphate Isomerases

    PubMed Central

    Cech, David; Wang, Pan Fen; Holler, Tod P.

    2014-01-01

    Arabinose-5-phosphate isomerases (APIs) catalyze the interconversion of d-ribulose-5-phosphate and d-arabinose-5-phosphate, the first step in the biosynthesis of 3-deoxy-d-manno-octulosonic acid (Kdo), an essential component of the lipopolysaccharide in Gram-negative bacteria. Classical APIs, such as Escherichia coli KdsD, contain a sugar isomerase domain and a tandem cystathionine beta-synthase domain. Despite substantial effort, little is known about structure-function relationships in these APIs. We recently reported an API containing only a sugar isomerase domain. This protein, c3406 from E. coli CFT073, has no known physiological function. In this study, we investigated a putative single-domain API from the anaerobic Gram-negative bacterium Bacteroides fragilis. This putative API (UniProt ID Q5LIW1) is the only protein encoded by the B. fragilis genome with significant identity to any known API, suggesting that it is responsible for lipopolysaccharide biosynthesis in B. fragilis. We tested this hypothesis by preparing recombinant Q5LIW1 protein (here referred to by the UniProt ID Q5LIW1), characterizing its API activity in vitro, and demonstrating that the gene encoding Q5LIW1 (GenBank ID YP_209877.1) was able to complement an API-deficient E. coli strain. We demonstrated that Q5LIW1 is inhibited by cytidine 5′-monophospho-3-deoxy-d-manno-2-octulosonic acid, the final product of the Kdo biosynthesis pathway, with a Ki of 1.91 μM. These results support the assertion that Q5LIW1 is the API that supports lipopolysaccharide biosynthesis in B. fragilis and is subject to feedback regulation by CMP-Kdo. The sugar isomerase domain of E. coli KdsD, lacking the two cystathionine beta-synthase domains, demonstrated API activity and was further characterized. These results suggest that Q5LIW1 may be a suitable system to study API structure-function relationships. PMID:24891442

  14. Arabinose 5-phosphate isomerase as a target for antibacterial design: studies with substrate analogues and inhibitors.

    PubMed

    Gabrielli, Luca; Merlo, Silvia; Airoldi, Cristina; Sperandeo, Paola; Gianera, Serena; Polissi, Alessandra; Nicotra, Francesco; Holler, Tod P; Woodard, Ronald W; Cipolla, Laura

    2014-04-15

    Structural requirements of D-arabinose 5-phosphate isomerase (KdsD, E.C. 5.3.1.13) from Pseudomonas aeruginosa were analysed in detail using advanced NMR techniques. We performed epitope mapping studies of the binding between the enzyme and the most potent KdsD inhibitors found to date, together with studies of a set of newly synthesised arabinose 5-phosphate (A5P) mimetics. We report here the first experimental evidence that KdsD may bind the furanose form of A5P, suggesting that catalysis of ring opening may be an important part of KdsD catalysis. PMID:24680056

  15. Structure of escherichia coli ribose-5-phosphate isomerase : a ubiquitous enzyme of the pentose phosphate pathway and the Calvin cycle.

    SciTech Connect

    Zhang, R.; Andersson, C. E.; Savchenko, A.; Skarina, T.; Evdokimova, E.; Beasley, S.; Arrowsmith, C. H.; Edwards, A.; Joachimiak, A.; Mowbray, S. L.; Biosciences Division; Uppsala Univ.; Univ. Health Network; Univ. of Toronto; Swedish Univ. of Agricultural Sciences

    2003-01-01

    Ribose-5-phosphate isomerase A (RpiA; EC 5.3.1.6) interconverts ribose-5-phosphate and ribulose-5-phosphate. This enzyme plays essential roles in carbohydrate anabolism and catabolism; it is ubiquitous and highly conserved. The structure of RpiA from Escherichia coli was solved by multiwavelength anomalous diffraction (MAD) phasing, and refined to 1.5 Angstroms resolution (R factor 22.4%, R{sub free} 23.7%). RpiA exhibits an {alpha}/{beta}/({alpha}/{beta})/{beta}/{alpha} fold, some portions of which are similar to proteins of the alcohol dehydrogenase family. The two subunits of the dimer in the asymmetric unit have different conformations, representing the opening/closing of a cleft. Active site residues were identified in the cleft using sequence conservation, as well as the structure of a complex with the inhibitor arabinose-5-phosphate at 1.25 A resolution. A mechanism for acid-base catalysis is proposed.

  16. Utilization of plant hemicellulose for production of xylanase and glucose isomerase

    SciTech Connect

    Han, Y.W.

    1983-01-01

    The hemicellulose fraction of sugarcane bagasse and ryegrass straw was extracted with NaOH and used for production of xylanase and glucose isomerase by Streptomyces flavogriseus. As much as 33% of hemicellulose per dry weight of the plant material could be obtained by treating the plant biomass with 5-10% NaOH for 1 h at 121 degrees or for 24 h at room temperature. The amount of hemicellulose extracted greatly increased as the concentration of NaOH increased up to 10%, thereafter the rate of increase slowed. Hemicellulose was further fractionated into hemicellulose A and hemicellulose B. Growth of the organism on plant hemicellulose yielded glucose isomerase (2.2 units/mL culture) and xylanase (27 units/mL culture). The levels of the enzymes produced on hemicellulose were about the same as on xylose or xylan. The organism, however, grew poorly on sugarcane bagasse and produced little enzymes.

  17. Disclosing the essentiality of ribose-5-phosphate isomerase B in Trypanosomatids.

    PubMed

    Faria, Joana; Loureiro, Inês; Santarém, Nuno; Cecílio, Pedro; Macedo-Ribeiro, Sandra; Tavares, Joana; Cordeiro-da-Silva, Anabela

    2016-01-01

    Ribose-5-phosphate isomerase (RPI) belongs to the non-oxidative branch of the pentose phosphate pathway, catalysing the inter-conversion of D-ribose-5-phosphate and D-ribulose-5-phosphate. Trypanosomatids encode a type B RPI, whereas humans have a structurally unrelated type A, making RPIB worthy of exploration as a potential drug target. Null mutant generation in Leishmania infantum was only possible when an episomal copy of RPIB gene was provided, and the latter was retained both in vitro and in vivo in the absence of drug pressure. This suggests the gene is essential for parasite survival. Importantly, the inability to remove the second allele of RPIB gene in sKO mutants complemented with an episomal copy of RPIB carrying a mutation that abolishes isomerase activity suggests the essentiality is due to its metabolic function. In vitro, sKO promastigotes exhibited no defect in growth, metacyclogenesis or macrophage infection, however, an impairment in intracellular amastigotes' replication was observed. Additionally, mice infected with sKO mutants rescued by RPIB complementation had a reduced parasite burden in the liver. Likewise, Trypanosoma brucei is resistant to complete RPIB gene removal and mice infected with sKO mutants showed prolonged survival upon infection. Taken together our results genetically validate RPIB as a potential drug target in trypanosomatids. PMID:27230471

  18. Disclosing the essentiality of ribose-5-phosphate isomerase B in Trypanosomatids

    PubMed Central

    Faria, Joana; Loureiro, Inês; Santarém, Nuno; Cecílio, Pedro; Macedo-Ribeiro, Sandra; Tavares, Joana; Cordeiro-da-Silva, Anabela

    2016-01-01

    Ribose-5-phosphate isomerase (RPI) belongs to the non-oxidative branch of the pentose phosphate pathway, catalysing the inter-conversion of D-ribose-5-phosphate and D-ribulose-5-phosphate. Trypanosomatids encode a type B RPI, whereas humans have a structurally unrelated type A, making RPIB worthy of exploration as a potential drug target. Null mutant generation in Leishmania infantum was only possible when an episomal copy of RPIB gene was provided, and the latter was retained both in vitro and in vivo in the absence of drug pressure. This suggests the gene is essential for parasite survival. Importantly, the inability to remove the second allele of RPIB gene in sKO mutants complemented with an episomal copy of RPIB carrying a mutation that abolishes isomerase activity suggests the essentiality is due to its metabolic function. In vitro, sKO promastigotes exhibited no defect in growth, metacyclogenesis or macrophage infection, however, an impairment in intracellular amastigotes’ replication was observed. Additionally, mice infected with sKO mutants rescued by RPIB complementation had a reduced parasite burden in the liver. Likewise, Trypanosoma brucei is resistant to complete RPIB gene removal and mice infected with sKO mutants showed prolonged survival upon infection. Taken together our results genetically validate RPIB as a potential drug target in trypanosomatids. PMID:27230471

  19. Ribose-5-phosphate isomerase from Saccharomyces cerevisiae: purification and molecular analysis of the enzyme.

    PubMed

    Reuter, R; Naumann, M; Bär, J; Miosga, T; Kopperschläger, G

    1998-01-01

    Purification and molecular analysis of ribose-5-phosphate isomerase (EC 5.3.1.6) from Saccharomyces cerevisiae is described first time. The enzyme was enriched from a haploid deletion mutant containing the wild-type gene on a multicopy plasmid elaborating the following steps: ammonium sulphate precipitation, interfacial salting out on Sepharose 6B, high performance liquid chromatography on Fractogel EMD DEAE and on Resource Phenyl. The enzyme activity was found to be rather unstable possibly caused by removal of stabilizing cofactors or proteins during the purification procedure. The purified enzyme showed a hyperbolic dependence on the substrate ribose-5-phosphate with a K(m)-value of 1.6 +/- 0.3 mmol/l. For the native enzyme a molecular mass of 115 +/- 10 kDa was determined as found by saccharose density gradient centrifugation, sedimentation equilibrium analysis, size exclusion chromatography and polyacrylamide gel electrophoresis. Sodium dodecyl sulphate polyacrylamide gel electrophoresis and Western blotting revealed one band with a molecular mass of 31 +/- 2 kDa. Thus, the native enzyme is composed of four subunits of identical size. The molecular mass of the subunit and the identified N-terminal sequence of 33 amino acids fits well the 258 amino acid protein encoded by the S. cerevisiae RKI open reading frame, which was characterized previously only by increasing specific activities of ribose-5-phosphate isomerase in cells after cloning the gene. On the basis of the conserved amino acids an alignment of the amino acid sequence of ribose-5-phosphate isomerase from yeast with those of the enzyme from mouse, spinach and Escherichia coli is presented. PMID:9763699

  20. Concerted Proton Transfer Mechanism of Clostridium thermocellum Ribose-5-phosphate Isomerase

    PubMed Central

    Wang, Jun; Yang, Weitao

    2013-01-01

    Ribose-5-phosphate isomerase (Rpi) catalyzes the interconversion of D-ribose-5-phosphate and D-ribulose-5-phosphate and plays an essential role in the pentose phosphate pathway and the Calvin cycle of photosynthesis. RpiB, one of the two isoforms of Rpi, is also a potential drug target for some pathogenic bacteria. Clostridium thermocellum ribose-5-phosphate isomerase (CtRpi), belonging to RpiB family, has recently been employed to the industrial production of rare sugars because of it fast reactions kinetics and narrow substrate specificity. It is known this enzyme adopts proton transfer mechanism. It was suggested that the deprotonated Cys65 attracts the proton at C2 of substrate to initiate the isomerization reaction and this step is the rate-limiting step. However the elaborate catalytic mechanism is still unclear. We have performed quantum mechanical/molecular mechanical simulations of this rate-limiting step of the reaction catalyzed by CtRpi with the substrate D-ribose. Our results demonstrate that the deprotonated Cys65 is not a stable reactant. Instead, our calculations revealed a concerted proton-transfer mechanism: Asp8, a highly conserved residue in the RpiB family performs as the base to abstract the proton at Cys65 and Cys65 in turn abstracts the proton of the D-ribose simultaneously. Moreover, we found Thr67 cannot catalyze the proton transfer from O2 to O1 of the D-ribose alone. Water molecule(s) may assist this proton transfer with Thr67. Our findings lead to a clear understanding of the catalysis mechanism of RpiB family and should guide the experiments to increase the catalysis efficiency. This study also highlights the importance of initial protonation states of cysteines. PMID:23875675

  1. Glucose(xylose) isomerase production by Streptomyces sp. CH7 grown on agricultural residues.

    PubMed

    Chanitnun, Kankiya; Pinphanichakarn, Pairoh

    2012-07-01

    Streptomyces sp. CH7 was found to efficiently produce glucose(xylose) isomerase when grown on either xylan or agricultural residues. This strain produced a glucose(xylose) isomerase activity of roughly 1.8 U/mg of protein when it was grown in medium containing 1% xylose as a carbon source. Maximal enzymatic activities of about 5 and 3 U/mg were obtained when 1% xylan and 2.5% corn husks were used, respectively. The enzyme was purified from a mycelial extract to 16-fold purity with only two consecutive column chromatography steps using Macro-prep DEAE and Sephacryl-300, respectively. The approximate molecular weight of the purified enzyme is 170 kDa, and it has four identical subunits of 43.6 kDa as estimated by SDS-PAGE. Its K m values for glucose and xylose were found to be 258.96 and 82.77 mM, respectively, and its V max values are 32.42 and 63.64 μM/min/mg, respectively. The purified enzyme is optimally active at 85°C and pH 7.0. It is stable at pH 5.5-8.5 and at temperatures up to 60°C after 30 min. These findings indicate that glucose(xylose) isomerase from Streptomyces sp. CH7 has the potential for industrial applications, especially for high-fructose syrup production and bioethanol fermentation from hemicellulosic hydrolysates by Saccharomyces cerevisiae. PMID:24031932

  2. Structural characterization of a ribose-5-phosphate isomerase B from the pathogenic fungus Coccidioides immitis

    PubMed Central

    2011-01-01

    Background Ribose-5-phosphate isomerase is an enzyme that catalyzes the interconversion of ribose-5-phosphate and ribulose-5-phosphate. This family of enzymes naturally occurs in two distinct classes, RpiA and RpiB, which play an important role in the pentose phosphate pathway and nucleotide and co-factor biogenesis. Results Although RpiB occurs predominantly in bacteria, here we report crystal structures of a putative RpiB from the pathogenic fungus Coccidioides immitis. A 1.9 Å resolution apo structure was solved by combined molecular replacement and single wavelength anomalous dispersion (SAD) phasing using a crystal soaked briefly in a solution containing a high concentration of iodide ions. RpiB from C. immitis contains modest sequence and high structural homology to other known RpiB structures. A 1.8 Å resolution phosphate-bound structure demonstrates phosphate recognition and charge stabilization by a single positively charged residue whereas other members of this family use up to five positively charged residues to contact the phosphate of ribose-5-phosphate. A 1.7 Å resolution structure was obtained in which the catalytic base of C. immitis RpiB, Cys76, appears to form a weakly covalent bond with the central carbon of malonic acid with a bond distance of 2.2 Å. This interaction may mimic that formed by the suicide inhibitor iodoacetic acid with RpiB. Conclusion The C. immitis RpiB contains the same fold and similar features as other members of this class of enzymes such as a highly reactive active site cysteine residue, but utilizes a divergent phosphate recognition strategy and may recognize a different substrate altogether. PMID:21995815

  3. Ribose 5-phosphate isomerase inhibits LC3 processing and basal autophagy.

    PubMed

    Heintze, Jacob; Costa, Joana R; Weber, Melanie; Ketteler, Robin

    2016-09-01

    Autophagy and cellular metabolism are tightly linked processes, but how individual metabolic enzymes regulate the process of autophagy is not well understood. This study implicates ribose-5-phosphate isomerase (RPIA), a key regulator of the pentose phosphate pathway, in the control of autophagy. We used a dual gene deletion strategy, combining shRNA-mediated knockdown studies with CRISPR/Cas9 genome editing. Knockdown of RPIA by shRNA or genomic deletion by CRISPR/Cas9 genome editing, results in an increase of ATG4B-mediated LC3 processing and in the appearance of LC3-positive autophagosomes in cells. Increased LC3 processing upon knockdown of RPIA can be reversed by treatment with the antioxidant N-acetyl cysteine. The results are consistent with a model in which RPIA suppresses autophagy and LC3 processing by modulation of redox signaling. PMID:27328773

  4. Oxyanion hole-stabilized stereospecific isomerization in ribose-5-phosphate isomerase (Rpi).

    PubMed

    Hamada, Keisuke; Ago, Hideo; Sugahara, Mitsuaki; Nodake, Yuichi; Kuramitsu, Seiki; Miyano, Masashi

    2003-12-01

    Ribose-5-phosphate isomerase (Rpi) acts as a key enzyme in the oxidative and reductive pentose-phosphate pathways for the conversion of ribose-5-phosphate (R5P) to ribulose-5-phosphate and vice versa. We have determined the crystal structures of Rpi from Thermus thermophilus HB8 in complex with the open chain form of the substrate R5P and the open chain form of the C2 epimeric inhibitor arabinose-5-phosphate as well as the apo form at high resolution. The crystal structures of both complexes revealed that these ring-opened epimers are bound in the active site in a mirror symmetry binding mode. The O1 atoms are stabilized by an oxyanion hole composed of the backbone amide nitrogens in the conserved motif. In the structure of the Rpi.R5P complex, the conversion moiety O1-C1-C2-O2 in cis-configuration interacts with the carboxyl oxygens of Glu-108 in a water-excluded environment. Furthermore, the C2 hydroxyl group is presumed to be highly polarized by short hydrogen bonding with the side chain of Lys-99. R5P bound as the ring-opened reaction intermediate clarified the high stereoselectivity of the catalysis and is consistent with an aldose-ketose conversion by Rpi that proceeds via a cis-enediolate intermediate. PMID:13679361

  5. Trigonal crystals of glucose isomerase require thymol for their growth and stability

    NASA Astrophysics Data System (ADS)

    Chayen, N. E.; Lloyd, L. F.; Collyer, C. A.; Blow, D. M.

    1989-09-01

    The growth of trigonal bypyramidal crystals of Arthrobacter glucose isomerase is only possible in the presence of thymol at a concentration exceeding 2.5mM; at lower thymol concentrations rectangular and rhombic crystals are formed. When thymol is removed from the supernatant of trigonal crystals, they fall apart and recrystallise as rectangles or rhombs. The disordering can be reversed if thymol is reintroduced before the trigonal crystals have dispersed, but the other crystal forms are stable in thymol. During the disintegration of trigonal crystals the supernatant concentration of protein is increased.

  6. Psoriatic therapeutics and glucose-6-phosphate dehydrogenase.

    PubMed

    Cotton, D W; van Rossum, E

    1975-01-01

    The inhibitory effects of various agents on the enzyme glucose-6-phosphate dehydrogenase have been studied in vitro. Stress is laid on the calculation of kinetic parameters such as true K-I values. The most active inhibitor was methotrexate, closely followed by cGMP. The increase in inhibitory activity after incubation of methotrexate with liver slices is discussed. PMID:167665

  7. Coimmobilization of glucoamylase and glucose isomerase by molecular deposition technique for one-step conversion of dextrin to fructose.

    PubMed

    Ge, Y; Wang, Y; Zhou, H; Wang, S; Tong, Y; Li, W

    1999-01-01

    Glucose isomerase was immobilized by itself with adsorption and coimmobilized with glucoamylase by molecular deposition technique using macroporous trimethylamine polystyrene beads. Approximately 77.5% of the enzyme added was immobilized. The pH-activity curve of the immobilized glucose isomerase was broadened, resulting in 75% retention of its maximum activity at pH 6.2. The Km of the immobilized glucose isomerase was 1.28-fold that of the soluble one. When the two enzymes were immobilized together, the system was found capable of functioning at pH 6.0 to produce fructose from starch and dextrin. At this pH, the total fructose output of the coimmobilized enzyme system after 24 h was 1.9 times that of the free enzyme system. PMID:9987846

  8. Studies on the combined action of amylases and glucose isomerase on starch and its hydrolyzate. Part I. Production, extraction, purification and kinetic behavior of glucose isomerase.

    PubMed

    Attia, R M; Ghali, Y; Roushdi, M; Eldin, M A

    1980-06-01

    Glucose isomerase was produced from Streptomyces phaeochromogenes by aerobic fermentation at 28 degrees C for 24 hrs. The crude enzyme was obtained by disintegrating the harvested cells. It was found that ammonium sulphate at a saturation of 0.3-0.5 gave the maximum enzyme recovery (88.8%) from the crude extract, while acetone gave 66.2% at a concentration of 3/1 (V/V). On this basis the crude enzyme extract was purified following several steps as concentration, dialysis, precipitation with (NH4)2 SO4, then passing through column of Amberlite CG-50, and the eluate was treated with acetone to precipitate the enzyme. The kinetics behavior was studied and it was found that: optimum D-glucose concentration was 0.8 M, Km was 0.25 M, optimum pH was 7.0 and temperature was 70 degrees C. Magnesium at concentration of 0.07 M gave the maximum activity and its Km was 0.024 M. Antagonistic effects of Na+, Ca++ and Fe+++ in presence of 0.07 M of Mg++ were studied. Km and Vmax at different levels of Mg++ concentration were determined and no change in Km value was observed, while Vmax was affected. These findings indicate that the Mg++ combined with enzyme independently of the substrate. PMID:7424065

  9. 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

  10. 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

  11. Heterologous expression and biochemical characterization of glucose isomerase from Thermobifida fusca.

    PubMed

    Deng, Hui; Chen, Sheng; Wu, Dan; Chen, Jian; Wu, Jing

    2014-06-01

    Glucose isomerase (GIase) catalyzes the isomerization of D-glucose to D-fructose. The GIase from Thermobifida fusca WSH03-11 was expressed in Escherichia coli BL21(DE3), and the purified enzyme took the form of a tetramer in solution and displayed a pI value of 5.05. The temperature optimum of GIase was 80 °C and its half life was about 2 h at 80 °C or 15 h at 70 °C. The pH optimum of GIase was 10 and the enzyme retained 95 % activity over the pH range of 5-10 after incubating at 4 °C for 24 h. Kinetic studies showed that the K m and K cat values of the enzyme are 197 mM and 1,688 min(-1), respectively. The maximum conversion yield of glucose (45 %, w/v) to fructose of the enzyme was 53 % at pH 7.5 and 70 °C. The present study provides the basis for the industrial application of recombinant T. fusca GIase in the production of high fructose syrup. PMID:24317483

  12. Identification and characterization of a thermostable bifunctional enzyme with phosphomannose isomerase and sugar-1-phosphate nucleotidylyltransferase activities from a hyperthermophilic archaeon, Pyrococcus horikoshii OT3.

    PubMed

    Akutsu, Jun-ichi; Zhang, Zilian; Morita, Rihito; Kawarabayasi, Yutaka

    2015-11-01

    Mannosylglycerate is known as a compatible solute, and plays important roles for salinity adaptation and high temperature stability of microorganisms. In the gene cluster for the mannosylglycerate biosynthetic pathway predicted from the genomic data of Pyrococcus horikoshii OT3, the PH0925 protein was found as a putative bifunctional enzyme with phosphomannose isomerase (PMI) and mannose-1-phosphate guanylyltransferase (Man-1-P GTase) activities, which can synthesize GDP-mannose when accompanied by a phosphomannomutase/phosphoglucomutase (PMM/PGM) enzyme (PH0923). The recombinant PH0925 protein, expressed in E. coli, exhibited both expected PMI and Man-1-P GTase activities, as well as absolute thermostability; 95 °C was the optimum reaction temperature. According to the guanylyltransferase activity (GTase) of the PH0925 protein, it was found that the protein can catalyze glucose-1-phosphate (Glc-1-P) and glucosamine-1-phosphate (GlcN-1-P) in addition to Man-1-P. The analyses of C-terminus-truncated forms of the PH0925 protein indicated that sugar-1-phosphate nucleotidylyltransferase (Sugar-1-P NTase) activity was located in the region from the N-terminus to the 345th residue, and that the C-terminal 114 residue region of the PH0925 protein inhibited the Man-1-P GTase activity. Conversely, the PMI activity was abolished by deletion of the C-terminal 14 residues. This is the first report of a thermostable enzyme with both PMI and multiple Sugar-1-P NTase activities. PMID:26290359

  13. Isoprenoid biosynthesis as a target for antibacterial and antiparasitic drugs: phosphonohydroxamic acids as inhibitors of deoxyxylulose phosphate reducto-isomerase

    PubMed Central

    2004-01-01

    Isoprenoid biosynthesis via the methylerythritol phosphate pathway is a target against pathogenic bacteria and the malaria parasite Plasmodium falciparum. 4-(Hydroxyamino)-4-oxobutylphosphonic acid and 4-[hydroxy(methyl)amino]-4-oxobutyl phosphonic acid, two novel inhibitors of DXR (1-deoxy-D-xylulose 5-phosphate reducto-isomerase), the second enzyme of the pathway, have been synthesized and compared with fosmidomycin, the best known inhibitor of this enzyme. The latter phosphonohydroxamic acid showed a high inhibitory activity towards DXR, much like fosmidomycin, as well as significant antibacterial activity against Escherichia coli in tests on Petri dishes. PMID:15473867

  14. Characterization of stress and methylglyoxal inducible triose phosphate isomerase (OscTPI) from rice

    PubMed Central

    Sharma, Shweta; Mustafiz, Ananda; Singla-Pareek, Sneh L.; Shankar Srivastava, Prem; Sopory, Sudhir Kumar

    2012-01-01

    As compared with plant system, triose phosphate isomerase (TPI), a crucial enzyme of glycolysis, has been well studied in animals. In order to characterize TPI in plants, a full-length cDNA encoding OscTPI was cloned from rice and expressed in E. coli. The recombinant OscTPI was purified to homogeneity and it showed Km value of 0.1281 ± 0.025 µM, and the Vmax value of 138.7 ± 16 µmol min−1mg−1 which is comparable to the kinetic values studied in other plants. The OscTPI was found to be exclusively present in the cytoplasm when checked with the various methods. Functional assay showed that OscTPI could complement a TPI mutation in yeast. Real time PCR analysis revealed that OscTPI transcript level was regulated in response to various abiotic stresses. Interestingly, it was highly induced under different concentration of methylglyoxal (MG) stress in a concentration dependent manner. There was also a corresponding increase in the protein and the enzyme activity of OscTPI both in shoot and root tissues under MG stress. Our result shows that increases in MG leads to the increase in TPI which results in decrease of DHAP and consequently decrease in the level of toxic MG. PMID:22902706

  15. Optimization of Fermentation Medium for the Production of Glucose Isomerase Using Streptomyces sp. SB-P1

    PubMed Central

    Bhasin, Sheetal; Modi, H. A.

    2012-01-01

    The combination of medium ingredients has a profound influence on the metabolic pathways running in the microorganism which regulates the production of numerous metabolites. Glucose isomerase (GI), an enzyme with huge potential in the market, can isomerise glucose into fructose. GI is used widely for the production of High-Fructose Corn Syrup (HFCS). HFCS is used as a sweetener in food and pharmaceutical industries. Streptomyces are well-known producers of numerous enzymes including glucose isomerase. An array of 75 isolates was screened for the production of glucose isomerase. The isolate Streptomyces sp. SB-P1 was found to produce maximum amount of extracellular GI. Sucrose and raffinose among pure carbon sources and corn cob and wheat husk among crude agro residues were found to yield high enzyme titers. Potassium nitrate among pure nitrogen sources and soy residues among crude sources gave maximum production. Quantitative effect of carbon, nitrogen, and inducer on GI was also determined. Plackett-Burman design was used to study the effect of different medium ingredients. Sucrose and xylose as carbon sources and peptone and soy residues as nitrogen sources proved to be beneficial for GI production. PMID:22900192

  16. Dihydroxyacetone phosphate. Its structure and reactivity with α-glycerophosphate dehydrogenase, aldolase and triose phosphate isomerase and some possible metabolic implications

    PubMed Central

    Reynolds, S. J.; Yates, D. W.; Pogson, C. I.

    1971-01-01

    1. Dihydroxyacetone phosphate exists in neutral aqueous solution at 20°C as a mixture of keto, gem-diol and enolic forms in the ratio 55:44:1. 2. The three forms are freely interconvertible and rate constants for these reactions have been determined. 3. Keto-dihydroxyacetone phosphate is the primary reactive species in the reactions catalysed by α-glycerophosphate dehydrogenase, aldolase and triose phosphate isomerase. 4. The proportion of keto form to gem-diol forms of dihydroxyacetone phosphate is temperature-dependent. At 37°C, 83% is keto-dihydroxyacetone phosphate. 5. The enzymological and metabolic consequences of these results are discussed. ImagesFig. 1.Fig. 3.Fig. 4.Fig. 5.Fig. 6. PMID:4330197

  17. Identification of GutQ from Escherichia coli as a d-Arabinose 5-Phosphate Isomerase

    PubMed Central

    Meredith, Timothy C.; Woodard, Ronald W.

    2005-01-01

    The glucitol operon (gutAEBDMRQ) of Escherichia coli encodes a phosphoenolpyruvate:sugar phosphotransferase system that metabolizes the hexitol d-glucitol (sorbitol). The functions for all but the last gene, gutQ, have been previously assigned. The high sequence similarity between GutQ and KdsD, a d-arabinose 5-phosphate isomerase (API) from the 3-deoxy-d-manno-octulosonate (KDO)-lipopolysaccharide (LPS) biosynthetic pathway, suggested a putative activity, but its role within the context of the gut operon remained unclear. Accordingly, the enzyme was cloned, overexpressed, and characterized. Recombinant GutQ was shown to indeed be a second copy of API from the E. coli K-12 genome with biochemical properties similar to those of KdsD, catalyzing the reversible aldol-ketol isomerization between d-ribulose 5-phosphate (Ru5P) and d-arabinose 5-phosphate (A5P). Genomic disruptions of each API gene were constructed in E. coli K-12. TCM11[(ΔkdsD)] was capable of sustaining essential LPS synthesis at wild-type levels, indicating that GutQ functions as an API inside the cell. The gut operon remained inducible in TCM7[(ΔgutQ)], suggesting that GutQ is not directly involved in d-glucitol catabolism. The conditional mutant TCM15[(ΔgutQΔkdsD)] was dependent on exogenous A5P both for LPS synthesis/growth and for upregulation of the gut operon. The phenotype was suppressed by complementation in trans with a plasmid encoding a functional copy of GutQ or by increasing the amount of A5P in the medium. As there is no obvious obligatory role for GutQ in the metabolism of d-glucitol and there is no readily apparent link between d-glucitol metabolism and LPS biosynthesis, it is suggested that A5P is not only a building block for KDO biosynthesis but also may be a regulatory molecule involved in expression of the gut operon. PMID:16199563

  18. 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.

  19. Polymorphic Protein Crystal Growth: Influence of Hydration and Ions in Glucose Isomerase.

    PubMed

    Gillespie, C M; Asthagiri, D; Lenhoff, A M

    2014-01-01

    Crystal polymorphs of glucose isomerase were examined to characterize the properties and to quantify the energetics of protein crystal growth. Transitions of polymorph stability were measured in poly(ethylene glycol)/NaCl solutions, and one transition point was singled out for more detailed quantitative analysis. Single crystal x-ray diffraction was used to confirm space groups and identify complementary crystal structures. Crystal polymorph stability was found to depend on the NaCl concentration, with stability transitions requiring > 1 M NaCl combined with a low concentration of PEG. Both salting-in and salting-out behavior was observed and was found to differ for the two polymorphs. For NaCl concentrations above the observed polymorph transition, the increase in solubility of the less stable polymorph together with an increase in the osmotic second virial coefficient suggests that changes in protein hydration upon addition of salt may explain the experimental trends. A combination of atomistic and continuum models was employed to dissect this behavior. Molecular dynamics simulations of the solvent environment were interpreted using quasi-chemical theory to understand changes in protein hydration as a function of NaCl concentration. The results suggest that protein surface hydration and Na(+) binding may introduce steric barriers to contact formation, resulting in polymorph selection. PMID:24955067

  20. Crystallization and preliminary X-ray analysis of the isomerase domain of glucosamine-6-phosphate synthase from Candida albicans

    SciTech Connect

    Olchowy, Jaroslaw; Milewski, Slawomir

    2005-11-01

    The isomerase domain of glucosamine-6-phosphate synthase from C. albicans has been crystallized and X-ray diffraction data have been collected. Preliminary analysis of the data reveals the oligomeric structure of the eukaryotic synthase to be a ‘dimer’ of prokaryotic-like dimers. Glucosamine-6-phosphate synthase (EC 2.6.1.16) catalyses the first and practically irreversible step in the hexosamine metabolism pathway, the end product of which, uridine 5′-diphospho-N-acetyl d-glucosamine, is an essential substrate for assembly of the cell wall. The isomerase domain, consisting of residues 346–712 (42 kDa), of glucosamine-6-phosphate synthase from Candida albicans has been crystallized. X-ray analysis revealed that the crystals belonged to space group I4, with unit-cell parameters a = b = 149, c = 103 Å. Diffraction data were collected to 3.8 Å. Preliminary results from molecular replacement using the homologous bacterial monomer reveal that the asymmetric unit contains two monomers that resemble a bacterial dimer. The crystal lattice consists of pairs of such symmetry-related dimers forming elongated tetramers.

  1. Targeting bacterial membranes: NMR spectroscopy characterization of substrate recognition and binding requirements of D-arabinose-5-phosphate isomerase.

    PubMed

    Airoldi, Cristina; Sommaruga, Silvia; Merlo, Silvia; Sperandeo, Paola; Cipolla, Laura; Polissi, Alessandra; Nicotra, Francesco

    2010-02-01

    Lipopolysaccharide (LPS) is an essential component of the outer membrane of gram-negative bacteria and consists of three elements: lipid A, the core oligosaccharide, and the O-antigen. The inner-core region is highly conserved and contains at least one residue of 3-deoxy-D-manno-octulosonate (Kdo). Arabinose-5-phosphate isomerase (API) is an aldo-keto isomerase catalyzing the reversible isomerization of D-ribulose-5-phosphate (Ru5P) to D-arabinose-5-phosphate (A5P), the first step of Kdo biosynthesis. By exploiting saturation transfer difference (STD) NMR spectroscopy, the structural requirements necessary for API substrate recognition and binding were identified, with the aim of designing new API inhibitors. In addition, simple experimental conditions for the STD experiments to perform a fast, robust, and efficient screening of small libraries of potential API inhibitors, allowing the identification of new potential leads, were set up. Due to the essential role of API enzymes in LPS biosynthesis and gram-negative bacteria survival, by exploiting these data, a new generation of potent antibacterial drugs could be developed. PMID:20039350

  2. Accumulation of glucose 6-phosphate or fructose 6-phosphate is responsible for destabilization of glucose transporter mRNA in Escherichia coli.

    PubMed

    Morita, Teppei; El-Kazzaz, Waleed; Tanaka, Yuya; Inada, Toshifumi; Aiba, Hiroji

    2003-05-01

    Previously we found that a mutation in either pgi or pfkA, encoding phosphoglucose isomerase or phosphofructokinase A, respectively, facilitates degradation of the ptsG mRNA in an RNase E-dependent manner in Escherichia coli (1). In this study, we examined the effects of a series of glycolytic genes on the degradation of ptsG mRNA and how the mutations destabilize the ptsG mRNA. The conditional lethal mutation ts8 in fda, encoding fructose-1,6-P(2) aldolase just downstream of pfkA in the glycolytic pathway, caused the destabilization of ptsG mRNA at the nonpermissive temperature. Mutations in any other gene did not destabilize the ptsG mRNA; rather, they reduced the ptsG transcription mainly by affecting the cAMP level. The rapid degradation of ptsG mRNA in mutant strains was completely dependent upon the presence of glucose or any one of its compounds, which enter the Embden-Meyerhof glycolytic pathway before the block points. A significant increase in the intracellular glucose-6-P level was observed in the presence of glucose in the pgi strain. An overexpression of glucose-6-phosphate dehydrogenase eliminated both the accumulation and the degradation of ptsG mRNA in the pgi strain. In addition, accumulation of fructose-6-P led to the rapid degradation of ptsG mRNA in a pgi pfkA mutant strain lacking glucose-6-P. We conclude that the RNase E-dependent destabilization of ptsG mRNA occurs in response to accumulation of glucose-6-P or fructose-6-P. PMID:12578824

  3. Functional expression of Burkholderia cenocepacia xylose isomerase in yeast increases ethanol production from a glucose-xylose blend.

    PubMed

    de Figueiredo Vilela, Leonardo; de Mello, Vinicius Mattos; Reis, Viviane Castelo Branco; Bon, Elba Pinto da Silva; Gonçalves Torres, Fernando Araripe; Neves, Bianca Cruz; Eleutherio, Elis Cristina Araújo

    2013-01-01

    This study presents results regarding the successful cloning of the bacterial xylose isomerase gene (xylA) of Burkholderia cenocepacia and its functional expression in Saccharomyces cerevisiae. The recombinant yeast showed to be competent to efficiently produce ethanol from both glucose and xylose, which are the main sugars in lignocellulosic hydrolysates. The heterologous expression of the gene xylA enabled a laboratorial yeast strain to ferment xylose anaerobically, improving ethanol production from a fermentation medium containing a glucose-xylose blend similar to that found in sugar cane bagasse hydrolysates. The insertion of xylA caused a 5-fold increase in xylose consumption, and over a 1.5-fold increase in ethanol production and yield, in comparison to that showed by the WT strain, in 24h fermentations, where it was not detected accumulation of xylitol. These findings are encouraging for further studies concerning the expression of B. cenocepacia xylA in an industrial yeast strain. PMID:23186665

  4. 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.

  5. An autosomal glucose-6-phosphate dehydrogenase (hexose-6-phosphate dehydrogenase) polymorphism in human saliva.

    PubMed

    Tan, S G; Ashton, G C

    1976-01-01

    Glucose-6-phosphate dehydrogenase (hexose-6-phosphate dehydrogenase) from human saliva has been demonstrated by the zymogram technique. Three phenotypes were found. Family and population studies suggested that these phenotypes are the products of an autosomal locus with two alleles Sgd-1 and Sgd-2. PMID:950237

  6. Extraction of hemicellulose from ryegrass straw for the production of glucose isomerase and use of the resulting straw residue for animal feed

    SciTech Connect

    Chen, W.P.; Anderson, A.W.

    1980-03-01

    The hemicellulose fraction of ryegrass straw was extracted with NaOH and used for the production of glucose isomerase by Streptomyces flavogriseus. The level of hemicellulose extracted increased proportionately with increasing NaOH concentration up to about 4%, then the rate of increase slowed down. Hemicellulose extraction was facilitated by the combined application of heat and NaOH. Approximately 15% hemicellulose (12% as pentosan) could be obtained by treating straw with 4% NaOH for either 3 hours at 90/sup 0/C or 24 hour at room temperature. The highest level (3.04 units/ml culture) of intracellular glucose isomerase was obtained when the organism was grown at 30 degrees Centigrade for two days on 2% straw hemicellulose. The organism also produced a high yield of glucose isomerase on xylose or xylan. The NaOH treated straw residue, after removal of hemicellulose, had approximately 75% higher digestibility and 20% higher feed efficiency for weanling meadow voles than untreated straw, but almost the equivalent to that obtained by NaOH treatment without removal of the hemicellulose. Thus, the residue could be used as animal feed. A process for the production of glucose isomerase and animal feed from ryegrass straw was also proposed.

  7. Biochemical mechanisms of glucose-6-phosphate dehydrogenase deficiency.

    PubMed Central

    Morelli, A; Benatti, U; Gaetani, G F; De Flora, A

    1978-01-01

    A solid-phase radioimmunoassay for human glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP+ 1-oxidoreductase; EC 1.1.1.49) was developed that allowed the specific activity of this enzyme protein to be measured in lysates from whole erythrocyte populations, in lysates from erythrocytes of different ages, and in purified samples. The enzyme was highly purified from erythrocytes of single donors by a simple procedure of affinity chromatography with insolubilized adenosine 2',5'-bisphosphate. These techniques were used in an attempt to elucidate the molecular mechanisms leading to deficiency of glucose-6-phosphate dehydrogenase activity in two genetic variants of the enzyme, i.e., the Mediterranean and the Seattle-like variants. The results indicate that the lowered activity of erythrocytes containing the Mediterranean variant of glucose-6-phosphate dehydrogenase is related to an enhanced rate of degradation of a catalytically defective protein synthesized at a nearly normal rate. Synthesis of a normally functioning protein and an increased breakdown of it are involved in the Seattle-like variant of the enzyme. Images PMID:273924

  8. Enhancing the flux of D-glucose to the pentose phosphate pathway in Saccharomyces cerevisiae for the production of D-ribose and ribitol.

    PubMed

    Toivari, Mervi H; Maaheimo, Hannu; Penttilä, Merja; Ruohonen, Laura

    2010-01-01

    Phosphoglucose isomerase-deficient (pgi1) strains of Saccharomyces cerevisiae were studied for the production of D-ribose and ribitol from D-glucose via the intermediates of the pentose phosphate pathway. Overexpression of the genes coding for NAD(+)-specific glutamate dehydrogenase (GDH2) of S. cerevisiae or NADPH-utilising glyceraldehyde-3-phosphate dehydrogenase (gapB) of Bacillus subtilis enabled growth of the pgi1 mutant strains on D-glucose. Overexpression of the gene encoding sugar phosphate phosphatase (DOG1) of S. cerevisiae was needed for the production of D-ribose and ribitol; however, it reduced the growth of the pgi1 strains expressing GDH2 or gapB in the presence of higher D-glucose concentrations. The CEN.PK2-1D laboratory strain expressing both gapB and DOG1 produced approximately 0.4 g l(-1) of D-ribose and ribitol when grown on 20 g l(-1) (w/v) D-fructose with 4 g l(-1) (w/v) D-glucose. Nuclear magnetic resonance measurements of the cells grown with (13)C-labelled D-glucose showed that about 60% of the D-ribose produced was derived from D-glucose. Strains deficient in both phosphoglucose isomerase and transketolase activities, and expressing DOG1 and GDH2 tolerated only low D-glucose concentrations (< or =2 g l(-1) (w/v)), but produced 1 g l(-1) (w/v) D-ribose and ribitol when grown on 20 g l(-1) (w/v) D-fructose with 2 g l(-1) (w/v) D-glucose. PMID:19711072

  9. 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...

  10. 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...

  11. Thermophilic Thermotoga maritima ribose-5-phosphate isomerase RpiB: optimized heat treatment purification and basic characterization.

    PubMed

    Sun, Fangfang; Zhang, Xiao-Zhou; Myung, Suwan; Zhang, Y-H Percival

    2012-04-01

    The open reading frame TM1080 from Thermotoga maritima encoding ribose-5-phosphate isomerase type B (RpiB) was cloned and over-expressed in Escherichia coli BL21 (DE3). After optimization of cell culture conditions, more than 30% of intracellular proteins were soluble recombinant RpiB. High-purity RpiB was obtained by heat pretreatment through its optimization in buffer choice, buffer pH, as well as temperature and duration of pretreatment. This enzyme had the maximum activity at 70°C and pH 6.5-8.0. Under its suboptimal conditions (60°C and pH 7.0), k(cat) and K(m) values were 540s(-1) and 7.6mM, respectively; it had a half lifetime of 71h, resulting in its turn-over number of more than 2×10(8)mol of product per mol of enzyme. This study suggests that it is highly feasible to discover thermostable enzymes from exploding genomic DNA database of extremophiles with the desired stability suitable for in vitro synthetic biology projects and produce high-purity thermoenzymes at very low costs. PMID:22333529

  12. Chemical- and thermal-induced unfolding of Leishmania donovani ribose-5-phosphate isomerase B: a single-tryptophan protein.

    PubMed

    Kaur, Preet Kamal; Supin, Jakka S; Rashmi, S; Singh, Sushma

    2014-08-01

    Ribose-5-phosphate isomerase B (RpiB), a crucial enzyme of pentose phosphate pathway, was proposed to be a potential drug target for visceral leishmaniasis. In this study, we have analyzed the biophysical properties of Leishmania donovani RpiB (LdRpiB) enzyme to gain insight into its unfolding pathway under various chemical and thermal denaturation conditions by using fluorescence and CD spectroscopy. LdRpiB inactivation precedes the structural transition at lower concentrations of both urea and guanidine hydrochloride (GdHCl). 8-Anilinonapthalene 1-sulfonic (ANS) binding experiments revealed the presence of molten globule intermediate at 1.5 M GdHCl and a nonnative intermediate state at 6-M urea concentration. Acrylamide quenching experiments further validated the above findings, as solvent accessibility of tryptophan residues increased with increase in GdHCl and urea concentration. The recombinant LdRpiB was completely unfolded at 6 M GdHCl, whereas the enzyme molecule was resistant to complete unfolding even at 8-M urea concentration. The GdHCl- and urea-mediated unfolding involves a three-state transition process. Thermal-induced denaturation revealed complete loss of enzyme activity at 65 °C with only 20 % secondary structure loss. The formation of the well-ordered β-sheet structures of amyloid fibrils was observed after 55 °C which increased linearly till 85 °C as detected by thioflavin T dye. This study depicts the stability of the enzyme in the presence of chemical and thermal denaturants and stability-activity relationship of the enzyme. The presence of the intermediate states may have major implications in the way the enzyme binds to its natural ligand under various conditions. Also, the present study provides insights into the properties of intermediate entities of this important enzyme. PMID:24907042

  13. Ceramide 1-phosphate stimulates glucose uptake in macrophages

    PubMed Central

    Ouro, Alberto; Arana, Lide; Gangoiti, Patricia; Rivera, Io-Guané; Ordoñez, Marta; Trueba, Miguel; Lankalapalli, Ravi S.; Bittman, Robert; Gomez-Muñoz, Antonio

    2014-01-01

    It is well established that ceramide 1-phosphate (C1P) is mitogenic and antiapoptotic, and that it is implicated in the regulation of macrophage migration. These activities require high energy levels to be available in cells. Macrophages obtain most of their energy from glucose. In this work, we demonstrate that C1P enhances glucose uptake in RAW264.7 macrophages. The major glucose transporter involved in this action was found to be GLUT 3, as determined by measuring its translocation from the cytosol to the plasma membrane. C1P-stimulated glucose uptake was blocked by selective inhibitors of phosphatidylinositol 3-kinase (PI3K) or Akt, also known as protein kinase B (PKB), and by specific siRNAs to silence the genes encoding for these kinases. C1P-stimulated glucose uptake was also inhibited by pertussis toxin (PTX) and by the siRNA that inhibited GLUT 3 expression. C1P increased the affinity of the glucose transporter for its substrate, and enhanced glucose metabolism to produce ATP. The latter action was also inhibited by PI3K- and Akt-selective inhibitors, PTX, or by specific siRNAs to inhibit GLUT 3 expression. PMID:23333242

  14. Nomenclature of glucose-6-phosphate dehydrogenase in man*

    PubMed Central

    1967-01-01

    The World Health Organization convened in Geneva from 5 to 10 December 1966 a Scientific Group on Standardization of Procedures for the Study of Glucose-6-Phosphate Dehydrogenase1 (EC 1.1.1.49; D-glucose-6-phosphate: NAPD oxidoreductase; G6PD). Variants of this enzyme have attracted international attention both as causes of various haemolytic disorders and as useful genetic markers in man. In the course of the meeting the variants of this enzyme thus far described were extensively reviewed. There was unanimous agreement that a consistent system of nomenclature would be desirable, and that as G6PD variants were only one example of similar polymorphisms in man, a nomenclature should be devised which might conceivably be applied to other enzymes. The Group included the following recommendations on nomenclature in its report, which will be published in full in World Health Organization: Technical Report Series, 1967, 366. PMID:5299754

  15. Malaria, favism and glucose-6-phosphate dehydrogenase deficiency.

    PubMed

    Huheey, J E; Martin, D L

    1975-10-15

    Although glucose-6-phosphate dehydrogenase deficient individuals may suffer (sometimes fatally) from favism, a high incidence of this trait occurs in many Mediterranean populations. This apparent paradox is explained on the basis of a synergistic interaction between favism and G-6-PD deficiency that provides increased protection against malaria compared to that of the G-6-PD deficiency alone. This relationship is analogous to that between various hemoglobins and malaria in that there is selection for a more severe trait if it provides more protection against malaria. PMID:1107056

  16. l-Arabinose Isomerase and d-Xylose Isomerase from Lactobacillus reuteri: Characterization, Coexpression in the Food Grade Host Lactobacillus plantarum, and Application in the Conversion of d-Galactose and d-Glucose

    PubMed Central

    2014-01-01

    The l-arabinose isomerase (l-AI) and the d-xylose isomerase (d-XI) encoding genes from Lactobacillus reuteri (DSMZ 17509) were cloned and overexpressed in Escherichia coli BL21 (DE3). The proteins were purified to homogeneity by one-step affinity chromatography and characterized biochemically. l-AI displayed maximum activity at 65 °C and pH 6.0, whereas d-XI showed maximum activity at 65 °C and pH 5.0. Both enzymes require divalent metal ions. The genes were also ligated into the inducible lactobacillal expression vectors pSIP409 and pSIP609, the latter containing a food grade auxotrophy marker instead of an antibiotic resistance marker, and the l-AI- and d-XI-encoding sequences/genes were coexpressed in the food grade host Lactobacillus plantarum. The recombinant enzymes were tested for applications in carbohydrate conversion reactions of industrial relevance. The purified l-AI converted d-galactose to d-tagatose with a maximum conversion rate of 35%, and the d-XI isomerized d-glucose to d-fructose with a maximum conversion rate of 48% at 60 °C. PMID:24443973

  17. Erythrocyte glucose-6-phosphate dehydrogenase from Brazilian opossum Didelphis marsupialis.

    PubMed

    Barretto, O C de O; Oshiro, M; Oliveira, R A G; Fedullo, J D L; Nonoyama, K

    2006-05-01

    In a comparative study of erythrocyte metabolism of vertebrates, the specific activity of glucose-6-phosphate dehydrogenase (G6PD) of the Brazilian opossum Didelphis marsupialis in a hemolysate was shown to be high, 207 +/- 38 IU g-1 Hb-1 min-1 at 37 degrees C, compared to the human erythrocyte activity of 12 +/- 2 IU g-1 Hb-1 min-1 at 37 degrees C. The apparent high specific activity of the mixture led us to investigate the physicochemical properties of the opossum enzyme. We report that reduced glutathione (GSH) in the erythrocytes was only 50% higher than in human erythrocytes, a value lower than expected from the high G6PD activity since GSH is maintained in a reduced state by G6PD activity. The molecular mass, determined by G-200 Sephadex column chromatography at pH 8.0, was 265 kDa, which is essentially the same as that of human G6PD (260 kDa). The Michaelis-Menten constants (Km: 55 microM) for glucose-6-phosphate and nicotinamide adenine dinucleotide phosphate (Km: 3.3 microM) were similar to those of the human enzyme (Km: 50-70 and Km: 2.9-4.4, respectively). A 450-fold purification of the opossum enzyme was achieved and the specific activity of the purified enzyme, 90 IU/mg protein, was actually lower than the 150 IU/mg protein observed for human G6PD. We conclude that G6PD after purification from the hemolysate of D. marsupialis does not have a high specific activity. Thus, it is quite probable that the red cell hyperactivity reported may be explained by increased synthesis of G6PD molecules per unit of hemoglobin or to reduced inactivation in the RBC hemolysate. PMID:16648898

  18. Frostbite: A Novel Presentation of Glucose-6-Phosphate Dehydrogenase Deficiency?

    PubMed

    Bowles, Justin M; Joas, Chris; Head, Steven

    2015-01-01

    Acute hemolytic anemia (AHA) due to glucose 6-phosphate dehydrogenase (G6PD) deficiency has rarely been recognized as a contributor to the development of frostbite. We discuss a case of frostbite in a 32-year-old male Marine with G6PD deficiency during military training on Mount McKinley in Alaska, which eventually led to a permanent disability. In this report, the pathophysiology of G6PD deficiency, the effects of hemolytic anemia, and factors that contribute to frostbite will be discussed, as well as the clinical findings, treatment course, and the outcome of this case. The patient was evacuated and admitted to Alaska Regional Hospital. He was treated for fourth-degree frostbite, ultimately resulting in the complete or partial amputation of all toes. Although it cannot be proved that AHA occurred in this patient, this case potentially adds frostbite to the list of rare but possible clinical presentations of G6PD deficiency. PMID:26360347

  19. Kawasaki disease with Glucose-6-Phosphate Dehydrogenase deficiency, case report.

    PubMed

    Obeidat, Hesham Radi; Al-Dossary, Sahar; Asseri, Abdulsalam

    2015-09-01

    Kawasaki disease (KD) is an acute, self-limited vasculitis of unknown etiology that occurs predominantly in infants and children younger than 5 years of age. Coronary artery abnormalities are the most serious complication. Based on the literatures infusion of Intravenous Immunoglobulin of 2 g/kg and a high dose of oral aspirin up to 100 mg/kg/day are the standard treatment for Kawasaki disease in the acute stage, and should be followed by antiplatelet dose of aspirin for thrombocytosis. Glucose-6-Phosphate Dehydrogenase (G6PD) deficiency is an inherited X-linked hereditary disorder, and aspirin can induce hemolysis in patients with G6PD deficiency. We report a case of a 5 year and 8 month old male with KD and G6PD deficiency. PMID:27134550

  20. Multiple Independent Fusions of Glucose-6-Phosphate Dehydrogenase with Enzymes in the Pentose Phosphate Pathway

    PubMed Central

    Stover, Nicholas A.; Dixon, Thomas A.; Cavalcanti, Andre R. O.

    2011-01-01

    Fusions of the first two enzymes in the pentose phosphate pathway, glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconolactonase (6PGL), have been previously described in two distant clades, chordates and species of the malarial parasite Plasmodium. We have analyzed genome and expressed sequence data from a variety of organisms to identify the origins of these gene fusion events. Based on the orientation of the domains and range of species in which homologs can be found, the fusions appear to have occurred independently, near the base of the metazoan and apicomplexan lineages. Only one of the two metazoan paralogs of G6PD is fused, showing that the fusion occurred after a duplication event, which we have traced back to an ancestor of choanoflagellates and metazoans. The Plasmodium genes are known to contain a functionally important insertion that is not seen in the other apicomplexan fusions, highlighting this as a unique characteristic of this group. Surprisingly, our search revealed two additional fusion events, one that combined 6PGL and G6PD in an ancestor of the protozoan parasites Trichomonas and Giardia, and another fusing G6PD with phosphogluconate dehydrogenase (6PGD) in a species of diatoms. This study extends the range of species known to contain fusions in the pentose phosphate pathway to many new medically and economically important organisms. PMID:21829610

  1. Reduced neuronal expression of ribose-5-phosphate isomerase enhances tolerance to oxidative stress, extends lifespan, and attenuates polyglutamine toxicity in Drosophila

    PubMed Central

    Wang, Ching-Tzu; Chen, Yi-Chun; Wang, Yi-Yun; Huang, Ming-Hao; Yen, Tzu-Li; Li, Hsun; Liang, Cyong-Jhih; Sang, Tzu-Kang; Cho, Si-Chih; Yuh, Chiou-Hwa; Wang, Chao-Yung; Brummel, Theodore J.; Wang, Horng-Dar

    2011-01-01

    Summary Aging and age-related diseases can be viewed as the result of the lifelong accumulation of stress insults. The identification of mutant strains and genes which are responsive to stress and can alter longevity profiles provides new therapeutic targets for age-related diseases. Here we reported that a Drosophila strain with reduced expression of ribose-5-phosphate isomerase (rpi), EP2456, exhibits increased resistance to oxidative stress and enhanced lifespan. In addition, the strain also displays higher levels of NADPH. The knockdown of rpi in neurons by double-stranded RNA interference recapitulated the lifespan extension and oxidative stress resistance in Drosophila. This manipulation was also found to ameliorate the effects of genetic manipulations aimed at creating a model for studying Huntington’s disease by overexpression of polyglutamine in the eye, suggesting that modulating rpi levels could serve as a treatment for normal aging as well as for polyglutamine neurotoxicity. PMID:22040003

  2. Functional characterization of Helicobacter pylori 26695 sedoheptulose 7-phosphate isomerase encoded by hp0857 and its association with lipopolysaccharide biosynthesis and adhesion.

    PubMed

    Yu, Chung-Kai; Wang, Chun-Jen; Chew, Yongyu; Wang, Po-Chuan; Yin, Hsien-Sheng; Kao, Mou-Chieh

    2016-09-01

    Helicobacter pylori is a notorious human pathogen and the appearance of antibiotic resistance of this bacterium has posed a serious threat to human health. Lipopolysaccharide (LPS) is a key virulence factor and plays important roles in pathogenesis of H. pylori infection. Sedoheptulose 7-phosphate isomerase (GmhA), as an enzyme participating in the first step of heptose biosynthesis, is indispensable for the formation of inner core oligosaccharide of LPS. In this study, we cloned one putative gmhA ortholog, hp0857, from H. pylori 26695 and overexpressed it in Eschericha coli. Based on the results of molecular weight determination, the recombinant HP0857 is likely a homodimer. Analysis of enzymatic kinetic properties of this protein confirmed that hp0857 is indeed encoded a phosphoheptose isomerase which can utilize sedoheptulose 7-phosphate as the substrate in the ADP-L-glycero-D-manno-heptose (ADP- L,D-Hep) biosynthesis pathway. We also generated an HP0857 knockout mutant and explored its phenotypic changes. This mutant exhibited a decreased growth rate and displayed a "deep rough" type of LPS structure. In addition, it also had a slight decrease in its motility and was more susceptible to hydrophobic antibiotic novobiocin and detergents Triton X-100 and SDS. Furthermore, the adhesive capacity of the HP0857 knockout mutant to AGS cells was reduced significantly, and most of the infected cells didn't show a classic hummingbird phenotype. However, complementation of the HP0857 knockout mutation restored most of these phenotypic changes. In conclusion, we demonstrated that HP0857 protein is essential for inner core biosynthesis of H. pylori LPS and is a potential target for developing new antimicrobial agents against H. pylori infection. PMID:27369071

  3. Th1 stimulatory proteins of Leishmania donovani: comparative cellular and protective responses of rTriose phosphate isomerase, rProtein disulfide isomerase and rElongation factor-2 in combination with rHSP70 against visceral leishmaniasis.

    PubMed

    Jaiswal, Anil Kumar; Khare, Prashant; Joshi, Sumit; Kushawaha, Pramod Kumar; Sundar, Shyam; Dube, Anuradha

    2014-01-01

    In visceral leishmaniasis, the recovery from the disease is always associated with the generation of Th1-type of cellular responses. Based on this, we have previously identified several Th1-stimulatory proteins of Leishmania donovani -triose phosphate isomerase (TPI), protein disulfide isomerase (PDI) and elongation factor-2 (EL-2) etc. including heat shock protein 70 (HSP70) which induced Th1-type of cellular responses in both cured Leishmania patients/hamsters. Since, HSPs, being the logical targets for vaccines aimed at augmenting cellular immunity and can be early targets in the immune response against intracellular pathogens; they could be exploited as vaccine/adjuvant to induce long-term immunity more effectively. Therefore, in this study, we checked whether HSP70 can further enhance the immunogenicity and protective responses of the above said Th1-stimulatory proteins. Since, in most of the studies, immunogenicity of HSP70 of L. donovani was assessed in native condition, herein we generated recombinant HSP70 and tested its potential to stimulate immune responses in lymphocytes of cured Leishmania infected hamsters as well as in the peripheral blood mononuclear cells (PBMCs) of cured patients of VL either individually or in combination with above mentioned recombinant proteins. rLdHSP70 alone elicited strong cellular responses along with remarkable up-regulation of IFN-γ and IL-12 cytokines and extremely lower level of IL-4 and IL-10. Among the various combinations, rLdHSP70 + rLdPDI emerged as superior one augmenting improved cellular responses followed by rLdHSP70 + rLdEL-2. These combinations were further evaluated for its protective potential wherein rLdHSP70 + rLdPDI again conferred utmost protection (∼80%) followed by rLdHSP70 + rLdEL-2 (∼75%) and generated a strong cellular immune response with significant increase in the levels of iNOS transcript as well as IFN-γ and IL-12 cytokines which was further supported by the high level of IgG2 antibody

  4. Co-expression of D-glucose isomerase and D-psicose 3-epimerase: development of an efficient one-step production of D-psicose.

    PubMed

    Men, Yan; Zhu, Yueming; Zeng, Yan; Izumori, Ken; Sun, Yuanxia; Ma, Yanhe

    2014-10-01

    D-Psicose has been attracting attention in recent years because of its alimentary activities and is used as an ingredient in a range of foods and dietary supplements. To develop a one-step enzymatic process of D-psicose production, thermoactive D-glucose isomerase and the D-psicose 3-epimerase obtained from Bacillus sp. and Ruminococcus sp., respectively, were successfully co-expressed in Escherichia coli BL21 strain. The substrate of one-step enzymatic process was D-glucose. The co-expression system exhibited maximum activity at 65 °C and pH 7.0. Mg(2+) could enhance the output of D-psicose by 2.32 fold to 1.6 g/L from 10 g/L of D-glucose. When using high-fructose corn syrup (HFCS) as substrate, 135 g/L D-psicose was produced under optimum conditions. The mass ratio of D-glucose, D-fructose, and D-psicose was almost 3.0:2.7:1.0, when the reaction reached equilibrium after an 8h incubation time. This co-expression system approaching to produce D-psicose has potential application in food and beverage products, especially softdrinks. PMID:25152409

  5. Roles of triosephosphate isomerase and aerobic metabolism in Trypanosoma brucei.

    PubMed Central

    Helfert, S; Estévez, A M; Bakker, B; Michels, P; Clayton, C

    2001-01-01

    Kinetoplastid protozoa compartmentalize the first seven enzymes of glycolysis and two enzymes of glycerol metabolism in a microbody, the glycosome. While in its mammalian host, Trypanosoma brucei depends entirely on glucose for ATP generation. Under aerobic conditions, most of the glucose is metabolized to pyruvate. Aerobic metabolism depends on the activities of glycosomal triosephosphate isomerase and a mitochondrial glycerophosphate oxidase, and on glycerophosphate<-->dihydroxyacetone phosphate exchange across the glycosomal membrane. Using a combination of genetics and computer modelling, we show that triosephosphate isomerase is probably essential for bloodstream trypanosome survival, but not for the insect-dwelling procyclics, which preferentially use amino acids as an energy source. When the enzyme level decreased to about 15% of that of the wild-type, the growth rate was halved. Below this level, a lethal rise in dihydroxyacetone phosphate was predicted. Expression of cytosolic triosephosphate isomerase inhibited cell growth. Attempts to knockout the trypanosome alternative oxidase genes (which are needed for glycerophosphate oxidase activity) were unsuccessful, but when we lowered the level of the corresponding mRNA by expressing a homologous double-stranded RNA, oxygen consumption was reduced fourfold and the rate of trypanosome growth was halved. PMID:11415442

  6. Hypohidrotic ectodermal dysplasia associated with glucose-6-phosphate dehydrogenase deficiency.

    PubMed

    Ermertcan, Aylin Türel; Yaşar, Ali; Kayhan, Tuba Çelebı; Gülen, Hüseyin; Ertan, Pelin

    2011-09-01

    Hypohidrotic ectodermal dysplasia (HED) is a syndrome characterized by hypodontia, hypotrichosis, and partial or total ecrine sweat gland deficiency. The most prevalent form of HED is inherited as an X linked pattern. Glucose-6-phosphate dehydrogenase (G-6-PD) deficiency is an X-linked recessive disease, which leads to hemolytic anemia and jaundice. It is expressed in males, while heterozygous females are usually clinically normal. A 12-year-old boy with the complaints of hair and eyebrow disturbances, teeth disfigurement, decreased sweating, and xerosis presented to the outpatient clinic. Dermatological examination revealed sparse hair and eyebrows, conical-shaped teeth, xerosis, syndactylia, transverse grooves, and discoloration of nails. Laboratory findings indicated anemia. His 3-year-old sister also had sparse hair and eyebrows, xerosis, and syndactylia. We learned that the patient had a previous history of neonatal jaundice and a diagnosis of G-6-PD deficiency. Although it has been shown that loci of ectodermal dysplasia and G-6-PD deficiency genes are near to one another, we did not find any case study reporting on occurrence of these two genetic diseases together. With the aspect of this rare and interesting case, the relationship between HED and G-6-PD deficiency was defined. PMID:22028581

  7. Hypohidrotic Ectodermal Dysplasia Associated with Glucose-6-Phosphate Dehydrogenase Deficiency

    PubMed Central

    Yaşar, Ali; Kayhan, Tuba Çelebİ; Gülen, Hüseyin; Ertan, Pelin

    2011-01-01

    Hypohidrotic ectodermal dysplasia (HED) is a syndrome characterized by hypodontia, hypotrichosis, and partial or total ecrine sweat gland deficiency. The most prevalent form of HED is inherited as an X linked pattern. Glucose-6-phosphate dehydrogenase (G-6-PD) deficiency is an X-linked recessive disease, which leads to hemolytic anemia and jaundice. It is expressed in males, while heterozygous females are usually clinically normal. A 12-year-old boy with the complaints of hair and eyebrow disturbances, teeth disfigurement, decreased sweating, and xerosis presented to the outpatient clinic. Dermatological examination revealed sparse hair and eyebrows, conical-shaped teeth, xerosis, syndactylia, transverse grooves, and discoloration of nails. Laboratory findings indicated anemia. His 3-year-old sister also had sparse hair and eyebrows, xerosis, and syndactylia. We learned that the patient had a previous history of neonatal jaundice and a diagnosis of G-6-PD deficiency. Although it has been shown that loci of ectodermal dysplasia and G-6-PD deficiency genes are near to one another, we did not find any case study reporting on occurrence of these two genetic diseases together. With the aspect of this rare and interesting case, the relationship between HED and G-6-PD deficiency was defined. PMID:22028581

  8. 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

  9. 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.

  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. 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.

  12. Glucose-6-phosphate dehydrogenase deficiency in Nigerian children.

    PubMed

    Williams, Olatundun; Gbadero, Daniel; Edowhorhu, Grace; Brearley, Ann; Slusher, Tina; Lund, Troy C

    2013-01-01

    Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human enzymopathy and in Sub-Saharan Africa, is a significant cause of infection- and drug-induced hemolysis and neonatal jaundice. Our goals were to determine the prevalence of G6PD deficiency among Nigerian children of different ethnic backgrounds and to identify predictors of G6PD deficiency by analyzing vital signs and hematocrit and by asking screening questions about symptoms of hemolysis. We studied 1,122 children (561 males and 561 females) aged 1 month to 15 years. The mean age was 7.4 ± 3.2 years. Children of Yoruba ethnicity made up the largest group (77.5%) followed by those Igbo descent (10.6%) and those of Igede (10.2%) and Tiv (1.8%) ethnicity. G6PD status was determined using the fluorescent spot method. We found that the overall prevalence of G6PD deficiency was 15.3% (24.1% in males, 6.6% in females). Yoruba children had a higher prevalence (16.9%) than Igede (10.5%), Igbo (10.1%) and Tiv (5.0%) children. The odds of G6PD deficiency were 0.38 times as high in Igbo children compared to Yoruba children (p=0.0500). The odds for Igede and Tiv children were not significantly different from Yoruba children (p=0.7528 and 0.9789 respectively). Mean oxygen saturation, heart rate and hematocrit were not significantly different in G6PD deficient and G6PD sufficient children. The odds of being G6PD deficient were 2.1 times higher in children with scleral icterus than those without (p=0.0351). In conclusion, we determined the prevalence of G6PD deficiency in Nigerian sub-populations. The odds of G6PD deficiency were decreased in Igbo children compared to Yoruba children. There was no association between vital parameters or hematocrit and G6PD deficiency. We found that a history of scleral icterus may increase the odds of G6PD deficiency, but we did not exclude other common causes of icterus such as sickle cell disease or malarial infection. PMID:23874768

  13. Using intron sequence comparisons in the triose-phosphate isomerase gene to study the divergence of the fall armyworm host strains.

    PubMed

    Nagoshi, R N; Meagher, R L

    2016-06-01

    The noctuid moth Spodoptera frugiperda (the fall armyworm) is endemic to the Western Hemisphere and appears to be undergoing sympatric speciation to produce two subpopulations that differ in their choice of host plants. The 'rice strain' and 'corn strain' are morphologically indistinguishable, requiring the use of genetic markers for identification. Because fall armyworm is a major pest of corn and several other agricultural crops, characterizing the strains has important economic consequences. In this study, comparisons were made of the intron sequences from the triose-phosphate isomerase (Tpi) gene isolated from 85 fall armyworm specimens collected from two host plants. Sixteen new strain-specific haplotypes based on intron polymorphisms are described that can facilitate the characterization of fall armyworm populations associated with different host plants. Comparisons of genetic diversity within and between the strains provides evidence that the corn strain is undergoing active selection and supports the proposal of directional interstrain mating occurring in the wild. Comparisons of the polymorphisms indicate that each intron undergoes different patterns of mutation that in some cases corresponds to host plant preferences. The results confirm that intron sequence comparisons are an effective approach to study fall armyworm population genetics. PMID:26991678

  14. 'Super-perfect' enzymes: Structural stabilities and activities of recombinant triose phosphate isomerases from Pyrococcus furiosus and Thermococcus onnurineus produced in Escherichia coli.

    PubMed

    Sharma, Prerna; Guptasarma, Purnananda

    2015-05-01

    Triose phosphate isomerases (TIMs) are considered to be 'kinetically perfect' enzymes, limited in their activity only by the rates of diffusion of substrate and product molecules. Most studies conducted thus far have been on mesophile-derived TIMs. Here, we report studies of two extremophile-derived TIMs produced in Escherichia coli: (i) TonTIM, sourced from the genome of the thermophile archaeon, Thermococcus onnurineus, and (ii) PfuTIM, sourced from the genome of the hyperthermophile archaeon, Pyrococcus furiosus (PfuTIM). Although these enzymes are presumed to have evolved to function optimally at temperatures close to the boiling point of water, we find that TonTIM and PfuTIM display second-order rate-constants of activity (k(cat)/K(m) values) comparable to mesophile-derived TIMs, at 25 °C. At 90 °C, TonTIM and PfuTIM reach maximum velocities of reaction of ∼ 10(6)-10(7) μmol/s/mg, and display k(cat)/K(m) values in the range of ∼ 10(10)-10(11) M(-1) s(-1), which are three orders of magnitude higher than those reported for mesophile TIMs. Further, the two enzymes display no signs of having undergone any structural unfolding at 90 °C. Such enzymes could thus probably be called 'super-perfect' enzymes. PMID:25824038

  15. Mutational and Structural Analysis of Conserved Residues in Ribose-5-Phosphate Isomerase B from Leishmania donovani: Role in Substrate Recognition and Conformational Stability

    PubMed Central

    Kaur, Preet Kamal; Tripathi, Neha; Desale, Jayesh; Neelagiri, Soumya; Yadav, Shailendra; Bharatam, Prasad V.; Singh, Sushma

    2016-01-01

    Ribose-5-phosphate isomerase B from Leishmania donovani (LdRpiB) is one of the potential drug targets against visceral leishmaniasis. In the present study, we have targeted several conserved amino acids for mutational analysis (i.e. Cys69, His11, His102, His138, Asp45, Tyr46, Pro47 and Glu149) to gain crucial insights into their role in substrate binding, catalysis and conformational stability of the enzyme. All the eight LdRpiB variants were cloned, sequenced, expressed and purified. C69S, H102N, D45N and E149A mutants exhibited complete loss of enzyme activity indicating that they are indispensable for the enzyme activity. Kinetic parameters were altered in case of H138N, H11N and P47A variants; however Y46F exhibited similar kinetic behaviour as wild type. All the mutants except H138N exhibited altered protein structure as determined by CD and fluorescence spectral analysis. This data was supported by the atomic level details of the conformational changes and substrate binding using molecular dynamic simulations. LdRpiB also exhibited activity with D-form of various aldose substrates in the order of D-ribose > D-talose > D-allose > D-arabinose. Our study provides insights for better understanding of substrate enzyme interactions which can rationalize the process of drug design against parasite RpiB. PMID:26953696

  16. Structural modeling and docking studies of ribose 5-phosphate isomerase from Leishmania major and Homo sapiens: a comparative analysis for Leishmaniasis treatment.

    PubMed

    Capriles, Priscila V S Z; Baptista, Luiz Phillippe R; Guedes, Isabella A; Guimarães, Ana Carolina R; Custódio, Fabio L; Alves-Ferreira, Marcelo; Dardenne, Laurent E

    2015-02-01

    Leishmaniases are caused by protozoa of the genus Leishmania and are considered the second-highest cause of death worldwide by parasitic infection. The drugs available for treatment in humans are becoming ineffective mainly due to parasite resistance; therefore, it is extremely important to develop a new chemotherapy against these parasites. A crucial aspect of drug design development is the identification and characterization of novel molecular targets. In this work, through an in silico comparative analysis between the genomes of Leishmania major and Homo sapiens, the enzyme ribose 5-phosphate isomerase (R5PI) was indicated as a promising molecular target. R5PI is an important enzyme that acts in the pentose phosphate pathway and catalyzes the interconversion of d-ribose-5-phosphate (R5P) and d-ribulose-5-phosphate (5RP). R5PI activity is found in two analogous groups of enzymes called RpiA (found in H. sapiens) and RpiB (found in L. major). Here, we present the first report of the three-dimensional (3D) structures and active sites of RpiB from L. major (LmRpiB) and RpiA from H. sapiens (HsRpiA). Three-dimensional models were constructed by applying a hybrid methodology that combines comparative and ab initio modeling techniques, and the active site was characterized based on docking studies of the substrates R5P (furanose and ring-opened forms) and 5RP. Our comparative analyses show that these proteins are structural analogs and that distinct residues participate in the interconversion of R5P and 5RP. We propose two distinct reaction mechanisms for the reversible isomerization of R5P to 5RP, which is catalyzed by LmRpiB and HsRpiA. We expect that the present results will be important in guiding future molecular modeling studies to develop new drugs that are specially designed to inhibit the parasitic form of the enzyme without significant effects on the human analog. PMID:25528729

  17. Comparisons of mammalian Giardia duodenalis assemblages based on the β-giardin, glutamate dehydrogenase and triose phosphate isomerase genes.

    PubMed

    Scorza, Andrea V; Ballweber, Lora R; Tangtrongsup, Sahatchai; Panuska, Carla; Lappin, Michael R

    2012-10-26

    The objective of this study was to determine and compare the assemblages of Giardia duodenalis isolated from mammalian fecal samples using the β-giardin (bg), glutamate dehydrogenase (gdh) and triosephosphate isomerase (tpi) genes. A total of 202 samples, either submitted to the Veterinary Diagnostic Laboratory (Parasitology) at Colorado State University or part of ongoing research studies, were typed. A subset of 50 dog samples were also assessed by the tpi-D-specific primers. Of these, 183 were from dogs, 13 were from cats, two were from llamas, and one each was from a calf, an alpaca, a sheep, and a horse. The majority of the dogs (171 of 183 isolates) in this study were infected with only dog-adapted Assemblage C or D. The tpi-D-specific primers confirmed that 28 of the samples that typed as Assemblage D by the bg and gdh genes were also Assemblage D by the tpi-D-specific primers. Only 12 isolates were Assemblage A alone or Assemblage A and Assemblage C or D. Of the 13 cat isolates, seven were Assemblage F, two were Assemblage D, three were Assemblage A and 1 contained both Assemblages C and D. The calf isolate was Assemblage E (gdh, tpi) and the alpaca (bg, gdh), llamas (gdh), sheep (bg, gdh, tpi) and horse (tpi) isolates were all Assemblage A. When the assemblage could be determined for more than one gene, 91 of 117 dog isolates gave consistent results and 8 of 9 cat isolates gave consistent results. PMID:22652427

  18. Glucose-6-Phosphate Dehydrogenase and Its Deficiency in Mutants of Corynebacterium glutamicum

    PubMed Central

    Ihnen, Ernel D.; Demain, Arnold L.

    1969-01-01

    Corynebacterium glutamicum is a member of a group of taxonomically related glutamate-excreting bacteria which utilize glucose both by the Embden-Meyerhof and the pentose phosphate pathways, the latter sequence accounting for 10 to 38% of the glucose metabolized. Some of the properties of glucose-6-phosphate dehydrogenase in crude extracts of C. glutamicum were studied. The enzyme was rapidly inactivated by dilution in tris (hydroxymethyl)aminomethane-hydrochloride buffer. This inactivation was prevented by the presence of 0.45 m NaCl. Mg++ was required for enzyme activity, but Mn++, Ca++, Sr++, and Ba++ were equally effective. Growth of the organism under differing conditions did not markedly affect the specific activity of the enzyme. A generally applicable method for detecting colonies deficient in glucose-6-phosphate dehydrogenase was developed. Mutants so obtained were found to be auxotrophic for tryptophan. Upon reversion of the tryptophan requirement, the revertants still retained the property of glucose-6-phosphate dehydrogenase deficiency. Neither the mutants nor the revertants could grow as rapidly as the parent culture in glucose, in gluconate, or in a complex medium. PMID:5788701

  19. Allosteric regulation of the partitioning of glucose-1-phosphate between glycogen and trehalose biosynthesis in Mycobacterium tuberculosis

    PubMed Central

    Asención Diez, Matías D.; Demonte, Ana M.; Syson, Karl; Arias, Diego G.; Gorelik, Andrii; Guerrero, Sergio A.; Bornemann, Stephen; Iglesias, Alberto A.

    2015-01-01

    Background Mycobacterium tuberculosis is a pathogenic prokaryote adapted to survive in hostile environments. In this organism and other Gram-positive actinobacteria, the metabolic pathways of glycogen and trehalose are interconnected. Results In this work we show the production, purification and characterization of recombinant enzymes involved in the partitioning of glucose-1-phosphate between glycogen and trehalose in M. tuberculosis H37Rv, namely: ADP-glucose pyrophosphorylase, glycogen synthase, UDP-glucose pyrophosphorylase and trehalose-6-phosphate synthase. The substrate specificity, kinetic parameters and allosteric regulation of each enzyme were determined. ADP-glucose pyrophosphorylase was highly specific for ADP-glucose while trehalose-6-phosphate synthase used not only ADP-glucose but also UDP-glucose, albeit to a lesser extent. ADP-glucose pyrophosphorylase was allosterically activated primarily by phosphoenolpyruvate and glucose-6-phosphate, while the activity of trehalose-6-phosphate synthase was increased up to 2-fold by fructose-6-phosphate. None of the other two enzymes tested exhibited allosteric regulation. Conclusions Results give information about how the glucose-1-phosphate/ADP-glucose node is controlled after kinetic and regulatory properties of key enzymes for mycobacteria metabolism. General significance This work increases our understanding of oligo and polysaccharides metabolism in M. tuberculosis and reinforces the importance of the interconnection between glycogen and trehalose biosynthesis in this human pathogen. PMID:25277548

  20. Phosphonomethyl analogues of hexose phosphates.

    PubMed

    Webster, D; Jondorf, W R; Dixon, H B

    1976-05-01

    The analogue of fructose 1,6-bisphosphate in which the phosphate group, -O-PO3H2, on C-6 is replaced by the phosphonomethyl group, -CH2-PO3H2, was made enzymically from the corresponding analogue of 3-phosphoglycerate. It was a substrate for aldolase, which was used to form it, but not for fructose 1,6-bisphosphatase. It was hydrolysed chemically to yield the corresponding analogue of fructose 6-phosphate [i.e. 6-deoxy-6-(phosphonomethyl)-D-fructose, or, more strictly, 6,7-dideoxy-7-phosphono-D-arabino-2-heptulose]. This proved to be a substrate for the sequential actions of glucose 6-phosphate isomerase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. Thus seven out of the nine enzymes of the glycolytic and pentose phosphate pathways so far tested catalyse the reactions of the phosphonomethyl isosteres of their substrates. PMID:7247

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

    PubMed Central

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

    2010-01-01

    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. PMID:20382127

  2. Bioconversion of D-glucose to D-psicose with immobilized D-xylose isomerase and D-psicose 3-epimerase on Saccharomyces cerevisiae spores.

    PubMed

    Li, Zijie; Li, Yi; Duan, Shenglin; Liu, Jia; Yuan, Peng; Nakanishi, Hideki; Gao, Xiao-Dong

    2015-08-01

    Saccharomyces cerevisiae spores are dormant cells, which can tolerate various types of environmental stress. In our previous work, we successfully developed biological and chemical methods for enzyme immobilization based on the structures of S. cerevisiae spore wall. In this study, we employed biological and chemical approaches for the immobilization of D-xylose isomerase (XI) from Thermus thermophilus and D-psicose 3-epimerase (DPEase) from Agrobacterium tumefaciens with yeast spores, respectively. The enzymatic properties of both immobilized XI and DPEase were characterized and the immobilized enzymes exhibit higher thermostability, broader pH tolerance, and good repeatability compared with free enzymes. Furthermore, we established a two-step approach for the bioconversion of D-glucose to D-psicose using immobilized enzymes. To improve the conversion yield, a multi-pot strategy was adopted for D-psicose production by repeating the two-step process continually. As a result, the yield of D-psicose was obviously improved and the highest yield reached about 12.0 %. PMID:26065389

  3. Growth inhibition of Mycobacterium smegmatis by prodrugs of deoxyxylulose phosphate reducto-isomerase inhibitors, promising anti-mycobacterial agents.

    PubMed

    Ponaire, Sarah; Zinglé, Catherine; Tritsch, Denis; Grosdemange-Billiard, Catherine; Rohmer, Michel

    2012-05-01

    Since Mycobacterium tuberculosis sets up several multiple anti-tuberculosis drug resistance mechanisms, development of new drugs with innovative target is urgent. The methylerythritol phosphate pathway (MEP) involved in the biosynthesis of essential metabolites for the survival of mycobacteria, represents such a target. Fosmidomycin 1a and FR900098 1b, two inhibitors of DXR, do not affect the viability of M. tuberculosis cells, due to a lack of uptake. To overcome the absence of the mycobacterial cell wall crossing of these compounds, we synthesized and tested the inhibition potency of acyloxymethyl phosphonate esters as prodrugs of fosmidomycin 1a, FR900098 1b and their analogs 2a and 2b on Mycobacterium smegmatis. Only the prodrugs 4b-6b inhibit the bacterial growth and could be effective anti-mycobacterial agents. PMID:22405649

  4. The binding of 2-deoxy-D-glucose 6-phosphate to glycogen phosphorylase b: kinetic and crystallographic studies.

    PubMed

    Oikonomakos, N G; Zographos, S E; Johnson, L N; Papageorgiou, A C; Acharya, K R

    1995-12-15

    Kinetic and crystallographic studies have characterized the effect of 2-deoxy-glucose 6-phosphate on the catalytic and structural properties of glycogen phosphorylase b. Previous work on the binding of glucose 6-phosphate, a potent physiological inhibitor of the enzyme, to T state phosphorylase b in the crystal showed that the inhibitor binds at the allosteric site and induces substantial conformational changes that affect the subunit-subunit interface. The hydrogen-bond from the O-2 hydroxyl of glucose 6-phosphate to the main-chain oxygen of Val40' represents the only hydrogen bond from the sugar to the other subunit, and this interaction appears important for promoting a more "tensed" structure than native T state phosphorylase b. 2-Deoxy-glucose 6-phosphate acts competitively with both the activator AMP and the substrate glucose 1-phosphate, with Ki values of 0.53 mM and 1.23 mM, respectively. The binding of 2-deoxy-glucose 6-phosphate to T state glycogen phosphorylase b in the crystal, has been investigated and the complex phosphorylase b: 2-deoxy-glucose 6-phosphate has been refined to give a crystallographic R factor of 17.3%, for data between 8 A and 2.3 A. 2-Deoxy-glucose 6-phosphate binds at the allosteric site as the a anomer and adopts a different conformation compared to glucose 6-phosphate. The two conformations differ by 160 degrees in the torsion angle about the C-5-C-6 bond. The contacts from the phosphate group are essentially identical to those made by the phosphate of glucose 6-phosphate but the 2-deoxy glucosyl moiety binds in a quite different orientation compared to the glucosyl of glucose 6-phosphate. 2-Deoxy-glucose 6-phosphate can be accommodated in the allosteric site with very little change in the protein, while structural comparisons show that the phosphorylase b: 2-deoxy-glucose 6-phosphate complex structure is overall more similar to a glucose-like complex than to the Glc-6-P complex structure. PMID:7500360

  5. Multiplex PCR Targeting tpi (Triose Phosphate Isomerase), tcdA (Toxin A), and tcdB (Toxin B) Genes for Toxigenic Culture of Clostridium difficile

    PubMed Central

    Lemee, Ludovic; Dhalluin, Anne; Testelin, Sabrina; Mattrat, Marie-Andre; Maillard, Karine; Lemeland, Jean-François; Pons, Jean-Louis

    2004-01-01

    A multiplex PCR toxigenic culture approach was designed for simultaneous identification and toxigenic type characterization of Clostridium difficile isolates. Three pairs of primers were designed for the amplification of (i) a species-specific internal fragment of the tpi (triose phosphate isomerase) gene, (ii) an internal fragment of the tcdB (toxin B) gene, and (iii) an internal fragment of the tcdA (toxin A) gene allowing distinction between toxin A-positive, toxin B-positive (A+B+) strains and toxin A-negative, toxin B-positive (A−B+) variant strains. The reliability of the multiplex PCR was established by using a panel of 72 C. difficile strains including A+B+, A−B−, and A−B+ toxigenic types and 11 other Clostridium species type strains. The multiplex PCR assay was then included in a toxigenic culture approach for the detection, identification, and toxigenic type characterization of C. difficile in 1,343 consecutive human and animal stool samples. Overall, 111 (15.4%) of 721 human samples were positive for C. difficile; 67 (60.4%) of these samples contained A+B+ toxigenic isolates, and none of them contained A−B+ variant strains. Fifty (8%) of 622 animal samples contained C. difficile strains, which were toxigenic in 27 (54%) cases, including 1 A−B+ variant isolate. Eighty of the 721 human stool samples (37 positive and 43 negative for C. difficile culture) were comparatively tested by Premier Toxins A&B (Meridian Bioscience) and Triage C. difficile Panel (Biosite) immunoassays, the results of which were found concordant with toxigenic culture for 82.5 and 92.5% of the samples, respectively. The multiplex PCR toxigenic culture scheme described here allows combined diagnosis and toxigenic type characterization for human and animal C. difficile intestinal infections. PMID:15583303

  6. Combination treatment with fingolimod and a pathogenic antigen prevents relapse of glucose‐6‐phosphate isomerase peptide‐induced arthritis

    PubMed Central

    Yoshida, Yuya; Mikami, Norihisa; Matsushima, Yuki; Miyawaki, Mai; Endo, Hiroki; Banno, Rie; Tsuji, Takumi; Fujita, Tetsuro

    2016-01-01

    Introduction Combination treatment with fingolimod (FTY720) plus pathogenic antigen is thought to prevent glucose‐6‐phosphate isomerase (GPI)325‐339‐induced arthritis progression by effective induction of immune tolerance. Here, we examined the efficacy of this combination treatment on remission maintenance. Methods GPI325‐339‐induced arthritis mice were treated for 5 days with FTY720 (1.0 mg/kg, p.o.) alone, GPI325–339 (10 μg/mouse, i.v.) alone, or with the FTY720 plus GPI325‐339 combination. In some experiments, mice were resensitized with GPI325‐339. Results Following resensitization with GPI325‐339, combination‐treated mice exhibited neither severe relapse nor elevated lymphocyte infiltration in joints. Neither anti‐human nor mouse GPI325‐339 antibody levels were correlated with clinical symptoms. This suggests that combination treatment prevents relapse following resensitization via regulation of pathogenic antigen‐specific T cells. The proportion of regulatory T (Treg) cells in inguinal lymph nodes was increased post treatment in the FTY720 alone and FTY720 plus GPI325‐339 groups. In contrast, the proportion of glucocorticoid‐induced tumor necrosis factor receptor‐family‐related gene/protein (GITR)+ non‐Treg cells was increased only in combination‐treated mice. Furthermore, GITR+ non‐Treg cells, which were induced by the combination treatment in vivo, possess suppressive activity and high ability to produce interleukin (IL)‐10. Conclusion GITR+ non‐Treg cells might play a key role in relapse prevention following resensitization. Thus, this combination treatment might establish immune tolerance by induction of GITR+ non‐Treg cells. PMID:27621810

  7. Improvement and characterization of a hyperthermophilic glucose isomerase from Thermoanaerobacter ethanolicus and its application in production of high fructose corn syrup.

    PubMed

    Liu, Zhi-Qiang; Zheng, Wei; Huang, Jian-Feng; Jin, Li-Qun; Jia, Dong-Xu; Zhou, Hai-Yan; Xu, Jian-Miao; Liao, Cheng-Jun; Cheng, Xin-Ping; Mao, Bao-Xing; Zheng, Yu-Guo

    2015-08-01

    High fructose corn syrup (HFCS) is an alternative of liquid sweetener to sucrose that is isomerized by commercial glucose isomerase (GI). One-step production of 55 % HFCS by thermostable GI has been drawn more and more attentions. In this study, a new hyperthermophilic GI from Thermoanaerobacter ethanolicus CCSD1 (TEGI) was identified by genome mining, and then a 1317 bp fragment encoding the TEGI was synthesized and expressed in Escherichia coli BL21(DE3). To improve the activity of TEGI, two amino acid residues, Trp139 and Val186, around the active site and substrate-binding pocket based on the structural analysis and molecular docking were selected for site-directed mutagenesis. The specific activity of mutant TEGI-W139F/V186T was 2.3-fold and the value of k cat/K m was 1.86-fold as compared to the wild type TEGI, respectively. Thermostability of mutant TEGI-W139F/V186T at 90 °C for 24 h showed 1.21-fold extension than that of wild type TEGI. During the isomerization of glucose to fructose, the yield of fructose could maintain above 55.4 % by mutant TEGI-W139F/V186T as compared to 53.8 % by wild type TEGI at 90 °C. This study paved foundation for the production of 55 % HFCS using the thermostable TEGI. PMID:26077737

  8. The pentose phosphate pathway of glucose metabolism. Enzyme profiles and transient and steady-state content of intermediates of alternative pathways of glucose metabolism in Krebs ascites cells

    PubMed Central

    Gumaa, K. A.; McLean, Patricia

    1969-01-01

    1. The pentose phosphate pathway in Krebs ascites cells was investigated for regulatory reactions. For comparison, the glycolytic pathway was studied simultaneously. 2. Activities of the pentose phosphate pathway enzymes were low in contrast with those of the enzymes of glycolysis. The Km values of glucose 6-phosphate dehydrogenase for both substrate and cofactor were about four times the reported upper limit for the enzyme from normal tissues. Fructose 1,6-diphosphate and NADPH competitively inhibited 6-phosphogluconate dehydrogenase. 3. About 28% of the hexokinase activity was in the particulate fraction of the cells. The soluble enzyme was inhibited by fructose 1,6-diphosphate and ribose 5-phosphate, but not by 3-phosphoglycerate. The behaviour of the partially purified soluble enzyme in vitro in a system simulating the concentrations of ATP, glucose 6-phosphate and Pi found in vivo is reported. 4. Kinetics of metabolite accumulation during the transient state after the addition of glucose to the cells indicated two phases of glucose phosphorylation, an initial rapid phase followed abruptly by a slow phase extending into the steady state. 5. Of the pentose phosphate pathway intermediates, accumulation of 6-phosphogluconate, sedoheptulose 7-phosphate and fructose 6-phosphate paralleled the accumulation of glucose 6-phosphate. Erythrose 4-phosphate reached the steady-state concentration by 2min., whereas the pentose phosphates accumulated linearly. 6. The mass-action ratios of the pentose phosphate pathway reactions were calculated. The transketolase reaction was at equilibrium by 30sec. and then progressively shifted away from equilibrium towards the steady-state ratio. The glucose 6-phosphate dehydrogenase was far from equilibrium at all times. 7. Investigation of the flux of [14C]glucose carbon confirmed the existence of an operative pentose phosphate pathway in ascites cells, contributing 1% of the total flux in control cells and 10% in cells treated with

  9. Induction of fatty acid synthase and S14 gene expression by glucose, xylitol and dihydroxyacetone in cultured rat hepatocytes is closely correlated with glucose 6-phosphate concentrations.

    PubMed Central

    Mourrieras, F; Foufelle, F; Foretz, M; Morin, J; Bouche, S; Ferre, P

    1997-01-01

    It is now well established that the transcription of several genes belonging to the glycolytic and lipogenic pathway is stimulated in the presence of a high glucose concentration in adipocytes and hepatocytes. We have previously proposed that glucose 6-phosphate could be the signal metabolite that transduces the glucose effect. This proposal has recently been challenged and both an intermediate of the pentose phosphate pathway, xylulose 5-phosphate, and metabolites of the later part of glycolysis (3-phosphoglycerate and phosphoenolpyruvate) have been proposed. To discriminate between these possibilities, we have measured concomitantly, in primary cultures of adult rat hepatocytes, the expression of the fatty acid synthase (FAS) and S14 genes and the concentration of glucose metabolites. We have used various substrates entering at different steps of the glycolytic pathway (glucose, dihydroxyacetone) and the pentose phosphate pathway (xylitol). When compared with 5 mM glucose, 25 mM glucose induces a marked increase in both S14 and FAS gene expression, detectable as early as 2 h and peaking at 6 h. Increasing concentrations (1-5 mM) of xylitol and dihydroxyacetone in the presence of 5 mM glucose are also able to induce S14 and FAS gene expression progressively. Among the various glucose metabolites measured, glucose 6-phosphate, in contrast with xylulose 5-phosphate and metabolites of the lower part of glycolysis, is the only one that shows a clear-cut parallelism between its concentration and the degree of S14 and FAS gene expression. We conclude that glucose 6-phosphate is the most likely signal metabolite for the glucose-induced transcription of this group of genes. PMID:9291103

  10. 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

  11. 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...

  12. 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 assay. 864.7360 Section 864.7360 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and...

  13. 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 assay. 864.7360 Section 864.7360 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and...

  14. 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 assay. 864.7360 Section 864.7360 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and...

  15. Lycopene production in recombinant strains of Escherichia coli is improved by knockout of the central carbon metabolism gene coding for glucose-6-phosphate dehydrogenase.

    PubMed

    Zhou, Yan; Nambou, Komi; Wei, Liujing; Cao, Jingjing; Imanaka, Tadayuki; Hua, Qiang

    2013-12-01

    Genetic manipulation was undertaken in order to understand the mechanism involved in the heterologous synthesis of lycopene in Escherichia coli. Knockout of the central carbon metabolic gene zwf (glucose-6-phosphate dehydrogenase) resulted in the enhancement of lycopene production (above 130 % relative to control). The amplification and overexpression of rate-limiting steps encoded by idi (isopentenyl diphosphate isomerase), dxs (1-deoxyxylulose-5-phosphate synthase) and ispDF (4-diphosphocytidyl-2C-methyl-D-erythritol synthase and 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase) genes improved lycopene synthesis from 0.89 to 5.39 mg g(-1) DCW. The combination of central metabolic genes knockout with the amplification of MEP pathway genes yielded best amounts of lycopene (6.85-7.55 mg g(-1) DCW). Transcript profiling revealed that idi and dxs were up-regulated in the zwf knock-out strain, providing a plausible explanation for the increase in lycopene yield observed in this strain. An increase in precursor availability might also have contributed to the improved lycopene production. PMID:24062132

  16. 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. PMID:26116788

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

    PubMed Central

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

    1999-01-01

    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

  18. 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

  19. 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

  20. Molecular Analysis of the Gene Encoding F420-Dependent Glucose-6-Phosphate Dehydrogenase from Mycobacterium smegmatis

    PubMed Central

    Purwantini, Endang; Daniels, Lacy

    1998-01-01

    The gene fgd, which codes for F420-dependent glucose-6-phosphate dehydrogenase (FGD), was cloned from Mycobacterium smegmatis, and its sequence was determined and analyzed. A homolog of FGD which has a very high similarity to the M. smegmatis FGD-derived amino acid sequence was identified in Mycobacterium tuberculosis. FGD showed significant homology with F420-dependent N5,N10-methylene-tetrahydromethanopterin reductase (MER) from methanogenic archaea and with several hypothetical proteins from M. tuberculosis and Archaeoglobus fulgidus, but FGD showed no significant homology with NADP-dependent glucose-6-phosphate dehydrogenases. Multiple alignment of FGD and MER proteins revealed four conserved consensus sequences. Multiple alignment of FGD with the hypothetical proteins also revealed portions of the same conserved sequences. Moderately high levels of FGD were expressed in Escherichia coli BL21(DE3) carrying fgd in pBluescript. PMID:9555906

  1. Histochemical research on metabolic pathways of glucose in some species of Mollusca Gastropoda.

    PubMed

    Bolognani Fantin, A M; Bolognani, L; Ottaviani, E; Franchini, A

    1987-01-01

    The metabolic pathways of glucose were studied by histochemical reactions in some species of gastropods living in different habitats. The glycolytic pathway is histochemically indicated by positive results for glucose-6-phosphate isomerase, fructose-1,6-biphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, and D-lactate dehydrogenase. The enzymes of the Krebs cycle gave different responses: isocitrate dehydrogenase and L-malate dehydrogenase were positive, whilst succinate dehydrogenase was constantly negative. Malate synthetase activity was also demonstrated. Despite L-glutamate dehydrogenase is undetectable, the presence of transaminase indicates the gluconeogenetic route. Phosphoglucomutase and glucose-6-phosphate phosphatase appear also positive. The metabolic meaning of our results were discussed. PMID:3111150

  2. 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

  3. The suitability of saliva for detection of glucose-6-phosphate dehydrogenase deficiency.

    PubMed

    Beamont, A H; Miguel, A; Goos, C M; Vermeesch-Markslag, A M; Hermans, A; Vermorken, A J

    1988-01-01

    Saliva was investigated for its suitability as a biopsy tissue for the determination of glucose-6-phosphate dehydrogenase deficiency. It appears that there is a significant difference between the activity of the enzyme in patients and controls. However, some controls have very low values making discrimination between patients and controls using a qualitative method impossible. Glucose-6-phosphate dehydrogenase deficiency is a relevant clinical problem in many rural areas in developing countries. Existing methods for determination of the deficiency in blood and hair follicles do not meet the criteria necessary for their large scale introduction in the areas of the world that are concerned by the problem. The present study shows that saliva is not a suitable alternative. Between the three biopsy tissues compared: blood, hair follicles and saliva, hair follicles remain most attractive since their isolation hardly involves the risk of infection. A simplified method for the detection of glucose-6-phosphate dehydrogenase activity in hair follicles that would allow health service workers in the field to determine the carrier status of pregnant women might form the basis for a future kernicterus prevention programme. PMID:3221843

  4. 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.

  5. Ischaemic Priapism and Glucose-6-Phosphate Dehydrogenase Deficiency: A Mechanism of Increased Oxidative Stress?

    PubMed Central

    Morrison, BF; Thompson, EB; Shah, SD; Wharfe, GH

    2014-01-01

    ABSTRACT Ischaemic priapism is a devastating urological condition that has the potential to cause permanent erectile dysfunction. The disorder has been associated with numerous medical conditions and the use of pharmacotherapeutic agents. The aetiology is idiopathic in a number of cases. There are two prior case reports of the association of ischaemic priapism and glucose-6-phosphate dehydrogenase (G6PD) deficiency. We report on a third case of priapism associated with G6PD deficiency and review recently described molecular mechanisms of increased oxidative stress in the pathophysiology of ischaemic priapism. The case report of a 32-year old Afro-Caribbean male with his first episode of major ischaemic priapism is described. Screening for common causes of ischaemic priapism, including sickle cell disease was negative. Glucose-6-phosphate dehydrogenase deficiency was discovered on evaluation for priapism. Penile aspiration was performed and erectile function was good post treatment. Glucose-6-phosphate dehydrogenase deficiency is a cause for ischaemic priapism and should be a part of the screening process in idiopathic causes of the disorder. Increased oxidative stress occurs in G6PD deficiency and may lead to priapism. PMID:25803385

  6. Purification and cloning of a thermostable xylose (glucose) isomerase with an acidic pH optimum from Thermoanaerobacterium strain JW/SL-YS 489.

    PubMed Central

    Liu, S Y; Wiegel, J; Gherardini, F C

    1996-01-01

    An unusual xylose isomerase produced by Thermoanaerobacterium strain JW/SL-YS 489 was purified 28-fold to gel electrophoretic homogeneity, and the biochemical properties were determined. Its pH optimum distinguishes this enzyme from all other previously described xylose isomerases. The purified enzyme had maximal activity at pH 6.4 (60 degrees C) or pH 6.8 (80 degrees C) in a 30-min assay, an isoelectric point at 4.7, and an estimated native molecular mass of 200 kDa, with four identical subunits of 50 kDa. Like other xylose isomerases, this enzyme required Mn2+, Co2+, or Mg2+ for thermal stability (stable for 1 h at 82 degrees C in the absence of substrate) and isomerase activity, and it preferred xylose as a substrate. The gene encoding the xylose isomerase was cloned and expressed in Escherichia coli, and the complete nucleotide sequence was determined. Analysis of the sequence revealed an open reading frame of 1,317 bp that encoded a protein of 439 amino acid residues with a calculated molecular mass of 50 kDa. The biochemical properties of the cloned enzyme were the same as those of the native enzyme. Comparison of the deduced amino acid sequence with sequences of other xylose isomerases in the database showed that the enzyme had 98% homology with a xylose isomerase from a closely related bacterium, Thermoanaerobacterium saccharolyticum B6A-RI. In fact, only seven amino acid differences were detected between the two sequences, and the biochemical properties of the two enzymes, except for the pH optimum, are quite similar. Both enzymes had a temperature optimum at 80 degrees C, very similar isoelectric points (pH 4.7 for strain JW/SL-YS 489 and pH 4.8 for T. saccharolyticum B6A-RI), and slightly different thermostabilities (stable for 1 h at 80 and 85 degrees C, respectively). The obvious difference was the pH optimum (6.4 to 6.8 and 7.0 to 7.5, respectively). The fact that the pH optimum of the enzyme from strain JW/SL-YS 489 was the property that differed

  7. Endogenous substrates of sphingosine-dependent kinases (SDKs) are chaperone proteins: heat shock proteins, glucose-regulated proteins, protein disulfide isomerase, and calreticulin.

    PubMed

    Megidish, T; Takio, K; Titani, K; Iwabuchi, K; Hamaguchi, A; Igarashi, Y; Hakomori, S

    1999-03-16

    Protein kinases whose activity is detectable only in the presence of sphingosine (Sph) or N,N'-dimethyl-Sph (DMS), but not in the presence of 15 other sphingolipids, phospholipids, and glycerolipids tested (Megidish, T., et al. (1995) Biochem. Biophys. Res. Commun. 216, 739-747), have been termed "sphingosine-dependent kinases" (SDKs). We showed previously that a purified SDK (termed "SDK1") phosphorylates a specific Ser position of adapter/chaperone protein 14-3-3 isoforms beta, eta, and zeta but not tau or sigma (Megidish, T., et al. (1998) J. Biol. Chem. 273, 21834-45). In this study we found the following: (i) other SDKs with different substrate specificities are present in cytosolic and membrane extracts of mouse Balb/c 3T3 (A31) fibroblasts. (ii) The activation of these SDKs is specific to D-erythro-Sph and its N-methyl derivatives, the effect of L-threo-Sph or its N-methyl derivatives is minimal, and nonspecific cationic amphiphiles have no effect at all. An SDK separated as fractions "TN31-33" phosphorylated a 50 kDa substrate which was identified as calreticulin, as well as two endogenous substrates with molecular mass 58 and 55 kDa, both identified as protein disulfide isomerase (PDI). This SDK, which specifically phosphorylates calreticulin and PDI, both molecular chaperones found at high levels in endoplasmic reticulum, is tentatively termed "SDK2". Another SDK activity was copurified with glucose-regulated protein (GRP) and heat shock proteins (HSP). One GRP substrate had the same amino acid sequence as GRP94 (synonym: endoplasmin); another HSP substrate had the same amino acid sequence as mouse HSP86 or HSP84, the analogues of human HSP90. An SDK activity separated and present in "fraction 42" from Q-Sepharose chromatography specifically phosphorylated GRP105 (or GRP94) and HSP68 but did not phosphorylate PDI or 14-3-3. This SDK is clearly different from other SDKs in its substrate specificity and is tentatively termed "SDK3". Interestingly

  8. Codon optimization of xylA gene for recombinant glucose isomerase production in Pichia pastoris and fed-batch feeding strategies to fine-tune bioreactor performance.

    PubMed

    Ata, Özge; Boy, Erdem; Güneş, Hande; Çalık, Pınar

    2015-05-01

    The objectives of this work are the optimization of the codons of xylA gene from Thermus thermophilus to enhance the production of recombinant glucose isomerase (rGI) in P. pastoris and to investigate the effects of feeding strategies on rGI production. Codons of xylA gene from T. thermophilus were optimized, ca. 30 % of the codons were replaced with those with higher frequencies according to the codon usage bias of P. pastoris, codon optimization resulted in a 2.4-fold higher rGI activity. To fine-tune bioreactor performance, fed-batch bioreactor feeding strategies were designed as continuous exponential methanol feeding with pre-calculated feeding rate based on the pre-determined specific growth rate, and fed-batch methanol-stat feeding. Six feeding strategies were designed, as follows: (S1) continuous exponential methanol- and pulse- sorbitol feeding; (S2) continuous exponential methanol- and peptone- feeding; (S3) continuous exponential methanol- and pulse- mannitol feeding; (S4) continuous exponential methanol- and peptone- feeding and pulse-mannitol feeding; (S5) methanol-stat feeding by keeping methanol concentration at 5 g L(-1); and, (S6) methanol-stat feeding by keeping methanol concentration at 5 g L(-1) and pulse-mannitol feeding. The highest cell and rGI activity was attained as 117 g L(-1) at t = 66 h and 32530 U L(-1) at t = 53 h, in strategy-S5. The use of the co-substrate mannitol does not increase the rGI activity in methanol-stat feeding, where 4.1-fold lower rGI activity was obtained in strategy-S6. The overall cell yield on total substrate was determined at t = 53 h as 0.21 g g(-1) in S5 strategy. PMID:25492311

  9. [Mutations of Q20L and G247D improved the specific-activity and optimum pH of glucose isomerase].

    PubMed

    Zhu, G P; Luo, D; Cai, Y F; Zhu, X Y; Teng, M K; Wang, Y Z

    2000-07-01

    The mutants of Q20L and G247D of glucose isomerase (GI) were constructed by in vitro site-directed mutagenesis of GI gene with double-primersmethod. The recombinant plasmids pTKD-GIQ20L and pTKD-GIG247D were expressed in E. coli K38 strain. The comparison experiments of mutant enzymes with wild-type GI showed that: (1) the optimum temperature of GIQ20L was decreased by 5 degrees C. Its thermostability was only 78% half-time of the wild type. But its substrate affinity was enhanced. (2) The specific-activity of GIG247D was increased by 33%, and the optimum pH was lowered by 0.6 unit. However, the thermostability of GIG247D was decreased. We supposed, based on the above facts and 0.19 nm resolution crystal structure of SM33GI, that Gln20 locates between alpha 0-helix and alpha 1-helix, the substitution of hydrophobic side chain of Leu for hydrophilic side chain of Gln may enhance the hydrophobic interaction of the molecular surface, leading to the decrease of the stability and thermostability of GIQ20L. Gly247 which is the last amino acid of a beta-sheet from 242 to 247 residues locates in the active core of GI. After replacement, Asp247 which has strong negative electricity may change the electrostatic distribution and influence the charge transfer processes of the active core. So the specific-activity of GIG247D was increased. The introduced charge could alter the pKa of dissociable groups and make the optimum pH lower. In addition, the side chain of Asp247 seems to be very crowded in the surrounding space conformation and is easy to exclude with the other side chains, therefore influences the stability of beta-sheet. Furthermore, Asp247 is in the vicinity of the interface of subunits, so it could interfere with the stability of the interaction between subunits. Thus, the GIG247D decreased the thermostability of SM33GI. The higher enzyme activity and the lower optimum pH will be very useful for industrial production of GI. PMID:11051821

  10. Purification and properties of the cytoplasmic glucose-6-phosphate dehydrogenase from pea leaves.

    PubMed

    Fickenscher, K; Scheibe, R

    1986-06-01

    A method involving affinity chromatography on the yellow dye Remazol Brilliant Gelb GL to highly purify the cytoplasmic isoenzyme of glucose-6-phosphate dehydrogenase from pea shoots is described. Purification is at least 6000-fold. The specific activity of the purified enzyme is 185 mumol NADP reduced/min per mg protein. The preparation was free from any contamination of chloroplastic isoenzyme. The purified enzyme retains its activity in the presence of reducing agents which, in contrast, inactivate the chloroplast enzyme. The state of activity of the cytoplasmic and the chloroplastic isoenzyme in illuminated or darkened pea leaves was investigated using specific antibodies. While upon illumination the chloroplastic isoenzyme was inactivated by 80 to 90%, we could not find any change in activity of the cytoplasmic glucose-6-phosphate dehydrogenase. ATP, ADP, NAD, NADH, and various sugar phosphates do not inhibit the enzyme activity. Only NADPH is a strong competitive inhibitor with respect to NADP, suggesting that the enzyme is regulated by feedback inhibition by one of its products. Mg2+ ions have no influence on the activity of the enzyme. The molecular weight has found to be 240,000 for the native enzyme and 60,000 for the subunit. Throughout the purification procedure the enzyme was very unstable unless NADP was present in the buffer. PMID:3717951

  11. The fate of 14C in glucose 6-phosphate synthesized from [1-14C]Ribose 5-phosphate by enzymes of rat liver.

    PubMed Central

    Williams, J F; Clark, M G; Blackmore, P F

    1978-01-01

    1. Glucose 5-phosphate was synthesized from ribose 5-phosphate by an enzyme extract prepared from an acetone-dried powder of rat liver. Three rates of ribose 5-phosphate utilization were observed during incubation for 17 h. An analysis of intermediates and products formed throughout the incubation revealed that as much as 20% of the substrate carbon could not be accounted for. 2. With [1-14C]ribose 5-phosphate as substrate, the specific radioactivity of [14C]glucose 6-phosphate formed was determined at 1, 2, 5 and 30 min and 3, 8 and 17 h. It increased rapidly to 1.9-fold the initial specific radioactivity of [1-14C]ribose 5-phosphate at 3 h and then decreased to a value approximately equal to that of the substrate at 6 h, and finally at 17 h reached a value 0.8-fold that of the initial substrate [1-14C]ribose 5-phosphate. 3. The specific radioactivity of [14C]ribose 5-phosphate decreased to approx. 50% of its inital value during the first 3 h of the incubation and thereafter remained unchanged. 4. The distribution of 14C in the six carbon atoms of [14C]glucose 6-phosphate formed from [1-14C]ribose 5-phosphate at 1, 2, 5 and 30 min and 3, 8 and 17 h was determined. The early time intervals (1--30 min) were characterized by large amounts of 14C in C-2 and in C-6 and with C-1 and C-3 being unlabelled. In contrast, the later time intervals (3--17 h) were characterized by the appearance of 14C in C-1 and C-3 and decreasing amounts of 14C in C-2 and C-6. 5. It is concluded that neither the currently accepted reaction sequence for the non-oxidative pentose phosphate pathway nor the 'defined' pentose phosphate-cycle mechanism can be reconciled with the labelling patterns observed in glucose 6-phosphate formed during the inital 3 h of the incubation. PMID:728109

  12. Inhibition of glucose-6-phosphate dehydrogenase sensitizes cisplatin-resistant cells to death

    PubMed Central

    Catanzaro, Daniela; Gaude, Edoardo; Orso, Genny; Giordano, Carla; Guzzo, Giulia; Rasola, Andrea; Ragazzi, Eugenio; Caparrotta, Laura; Frezza, Christian; Montopoli, Monica

    2015-01-01

    The mechanisms of cisplatin resistance, one of the major limitations of current chemotherapy, has only partially been described. We previously demonstrated that cisplatin-resistant ovarian cancer cells (C13), are characterized by reduced mitochondrial activity and higher glucose-dependency when compared to the cisplatin-sensitive counterpart (2008). In this work we further characterized the role of metabolic transformation in cisplatin resistance. By using transmitochondrial hybrids we show that metabolic reprogramming of cisplatin-resistant cell is not caused by inherent mtDNA mutations. We also found that C13 cells not only present an increased glucose-uptake and consumption, but also exhibit increased expression and enzymatic activity of the Pentose Phosphate pathway (PPP) enzyme Glucose-6-Phosphate Dehydrogenase (G6PDH). Moreover, we show that cisplatin-resistant cells are more sensitive to G6PDH inhibition. Even if the metabolomic fingerprint of ovarian cancer cells remains to be further elucidated, these findings indicate that PPP offers innovative potential targets to overcome cisplatin resistance. PMID:26337086

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

    PubMed Central

    Maleki, Susan; Mærk, Mali; Valla, Svein

    2015-01-01

    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. PMID:25746989

  14. 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.

  15. Is glucose-6-phosphate dehydrogenase deficiency more prevalent in Carrion's disease endemic areas in Latin America?

    PubMed

    Mazulis, Fernando; Weilg, Claudia; Alva-Urcia, Carlos; Pons, Maria J; Del Valle Mendoza, Juana

    2015-12-01

    Glucose-6-phosphate dehydrogenase (G6PD) is a cytoplasmic enzyme with an important function in cell oxidative damage prevention. Erythrocytes have a predisposition towards oxidized environments due to their lack of mitochondria, giving G6PD a major role in its stability. G6PD deficiency (G6PDd) is the most common enzyme deficiency in humans; it affects approximately 400 million individuals worldwide. The overall G6PDd allele frequency across malaria endemic countries is estimated to be 8%, corresponding to approximately 220 million males and 133 million females. However, there are no reports on the prevalence of G6PDd in Andean communities where bartonellosis is prevalent. PMID:26706684

  16. Glucose-6-Phosphate Dehydrogenase Deficiency and the Need for a Novel Treatment to Prevent Kernicterus.

    PubMed

    Cunningham, Anna D; Hwang, Sunhee; Mochly-Rosen, Daria

    2016-06-01

    Hyperbilirubinemia occurs frequently in newborns, and in severe cases can progress to kernicterus and permanent developmental disorders. Glucose-6-phosphate dehydrogenase (G6PD) deficiency, one of the most common human enzymopathies, is a major risk factor for hyperbilirubinemia and greatly increases the risk of kernicterus even in the developed world. Therefore, a novel treatment for kernicterus is needed, especially for G6PD-deficient newborns. Oxidative stress is a hallmark of bilirubin toxicity in the brain. We propose that the activation of G6PD via a small molecule chaperone is a potential strategy to increase endogenous defense against bilirubin-induced oxidative stress and prevent kernicterus. PMID:27235212

  17. Improvement of the quantitative method for glucose determination using hexokinase and glucose 6-phosphate dehydrogenase.

    PubMed

    Ogawa, Z; Kanashima, M; Nishioka, H

    2001-05-01

    This paper has two aims. The first one is to point out the shortcomings of Food and Drug Administration's (FDA's) reference method for the measurement of glucose. We found that the quantity of enzyme used in the method recommended by the FDA was more than the exact quantity needed for accurate measurement. The use of exact quantity of enzyme is important to minimize the negative effects due to impurity and side reactions of enzymes. The second aim is to simulate the coupling enzyme reaction using computer. We have successfully established the exact quantity of enzyme needed in the assay through the computer simulation. The quantity of the enzyme was lesser than the that recommended by FDA, but the reaction ended at the same time as in the FDA method. In addition, optimum conditions and inhibitory effects of various reagents have also been successfully analyzed using computer. In conclusion, we suggest a reference method using computer simulation to determine the exact quantity of the coupling enzyme needed in the assay. PMID:11434388

  18. [Effect of temperature acclimation on glucose-6-phosphate dehydrogenase activity in various tissues of the Mullus barbatus ponticus].

    PubMed

    Rusinova, O S

    1997-01-01

    Acclimation of the Mullus barbatus ponticus to the temperature fall (from 16 to 8 degrees C) induces an increase in the activity of glucose-6-phosphate dehydrogenase both in summer and in autumn. These changes are more expressed in the liver and red muscle than in the white ones. The values of Km of glucose-6-phosphate (only in autumn) and NADP are also higher at 8 than at 16 degrees C. Actinomycin D does not prevent from cold activation of this enzyme in liver only in autumn, when the activity of glucose-6-phosphate dehydrogenase is 8-10 times higher than in summer, and in the red muscle in the both seasons, although this injection decreases the level of enzyme activity in this muscle. This injection does not change the values of Km for glucose-6-phosphate and NADP. It is supposed that the activation of glucose-6-phosphate dehydrogenase in the liver and red muscle during cold adaptation may be a result of change of substrate-binding ability and enzyme interaction with NADP without changes in the enzyme biosynthesis. PMID:9606826

  19. Glucose-6-phosphate dehydrogenase deficiency and Alzheimer's disease: Partners in crime? The hypothesis.

    PubMed

    Ulusu, N Nuray

    2015-08-01

    Alzheimer's disease is a multifaceted brain disorder which involves various coupled irreversible, progressive biochemical reactions that significantly reduce quality of life as well as the actual life expectancy. Aging, genetic predispositions, head trauma, diabetes, cardiovascular disease, deficiencies in insulin signaling, dysfunction of mitochondria-associated membranes, cerebrovascular changes, high cholesterol level, increased oxidative stress and free radical formation, DNA damage, disturbed energy metabolism, and synaptic dysfunction, high blood pressure, obesity, dietary habits, exercise, social engagement, and mental stress are noted among the risk factors of this disease. In this hypothesis review I would like to draw the attention on glucose-6-phosphate dehydrogenase deficiency and its relationship with Alzheimer's disease. This enzymopathy is the most common human congenital defect of metabolism and defined by decrease in NADPH+H(+) and reduced form of glutathione concentration and that might in turn, amplify oxidative stress due to essentiality of the enzyme. This most common enzymopathy may manifest itself in severe forms, however most of the individuals with this deficiency are not essentially symptomatic. To understand the sporadic Alzheimer's disease, the writer of this paper thinks that, looking into a crystal ball might not yield much of a benefit but glucose-6-phosphate dehydrogenase deficiency could effortlessly give some clues. PMID:26004559

  20. 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 +/.

  1. Plasmodium falciparum glucose-6-phosphate dehydrogenase 6-phosphogluconolactonase is a potential drug target.

    PubMed

    Allen, Stacey M; Lim, Erin E; Jortzik, Esther; Preuss, Janina; Chua, Hwa Huat; MacRae, James I; Rahlfs, Stefan; Haeussler, Kristina; Downton, Matthew T; McConville, Malcolm J; Becker, Katja; Ralph, Stuart A

    2015-10-01

    The malarial parasite Plasmodium falciparum is exposed to substantial redox challenges during its complex life cycle. In intraerythrocytic parasites, haemoglobin breakdown is a major source of reactive oxygen species. Deficiencies in human glucose-6-phosphate dehydrogenase, the initial enzyme in the pentose phosphate pathway (PPP), lead to a disturbed redox equilibrium in infected erythrocytes and partial protection against severe malaria. In P. falciparum, the first two reactions of the PPP are catalysed by the bifunctional enzyme glucose-6-phosphate dehydrogenase 6-phosphogluconolactonase (PfGluPho). This enzyme differs structurally from its human counterparts and represents a potential target for drugs. In the present study we used epitope tagging of endogenous PfGluPho to verify that the enzyme localises to the parasite cytosol. Furthermore, attempted double crossover disruption of the PfGluPho gene indicates that the enzyme is essential for the growth of blood stage parasites. As a further step towards targeting PfGluPho pharmacologically, ellagic acid was characterised as a potent PfGluPho inhibitor with an IC50 of 76 nM. Interestingly, pro-oxidative drugs or treatment of the parasites with H2O2 only slightly altered PfGluPho expression or activity under the conditions tested. Furthermore, metabolic profiling suggested that pro-oxidative drugs do not significantly perturb the abundance of PPP intermediates. These data indicate that PfGluPho is essential in asexual parasites, but that the oxidative arm of the PPP is not strongly regulated in response to oxidative challenge. PMID:26198663

  2. 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

  3. Identification of glucoselysine-6-phosphate deglycase, an enzyme involved in the metabolism of the fructation product glucoselysine

    PubMed Central

    2005-01-01

    The metabolism of the glycation product fructose-ϵ-lysine in Escherichia coli involves its ATP-dependent phosphorylation by a specific kinase (FrlD), followed by the conversion of fructoselysine 6-phosphate into glucose 6-phosphate and lysine by fructoselysine-6-phosphate deglycase (FrlB), which is distantly related to the isomerase domain of glucosamine-6-phosphate synthase. As shown in the present work, several bacterial operons comprise: (1) a homologue of fructoselysine-6-phosphate deglycase; (2) a second homologue of the isomerase domain of glucosamine-6-phosphate synthase, more closely related to it; and (3) components of a novel phosphotransferase system, but no FrlD homologue. The FrlB homologue (GfrF) and the closer glucosamine-6-phosphate synthase homologue (GfrE) encoded by an Enterococcus faecium operon were expressed in E. coli and purified. Similar to FrlB, GfrF catalysed the reversible conversion of fructoselysine 6-phosphate into glucose 6-phosphate and lysine. When incubated with fructose 6-phosphate and elevated concentrations of lysine, GfrE catalysed the formation of a compound identified as 2-ϵ-lysino-2-deoxy-6-phospho-glucose (glucoselysine 6-phosphate) by NMR. GfrE also catalysed the reciprocal conversion, i.e. the formation of fructose 6-phosphate (but not glucose 6-phosphate) from glucoselysine 6-phosphate. The equilibrium constant of this reaction (0.8 M) suggests that the enzyme serves to degrade glucoselysine 6-phosphate. In conclusion, GfrF and GfrE serve to metabolize glycation products formed from lysine and glucose (fructoselysine) or fructose (glucoselysine), via their 6-phospho derivatives. The latter are presumably formed by the putative phosphotransferase system encoded by gfrA–gfrD. The designation gfr (glycation and fructation product degradation) is proposed for this operon. This is the first description of an enzyme participating in the metabolism of fructation products. PMID:16153181

  4. Identification of glucoselysine-6-phosphate deglycase, an enzyme involved in the metabolism of the fructation product glucoselysine.

    PubMed

    Wiame, Elsa; Lamosa, Pedro; Santos, Helena; Van Schaftingen, Emile

    2005-12-01

    The metabolism of the glycation product fructose-epsilon-lysine in Escherichia coli involves its ATP-dependent phosphorylation by a specific kinase (FrlD), followed by the conversion of fructoselysine 6-phosphate into glucose 6-phosphate and lysine by fructoselysine-6-phosphate deglycase (FrlB), which is distantly related to the isomerase domain of glucosamine-6-phosphate synthase. As shown in the present work, several bacterial operons comprise: (1) a homologue of fructoselysine-6-phosphate deglycase; (2) a second homologue of the isomerase domain of glucosamine-6-phosphate synthase, more closely related to it; and (3) components of a novel phosphotransferase system, but no FrlD homologue. The FrlB homologue (GfrF) and the closer glucosamine-6-phosphate synthase homologue (GfrE) encoded by an Enterococcus faecium operon were expressed in E. coli and purified. Similar to FrlB, GfrF catalysed the reversible conversion of fructoselysine 6-phosphate into glucose 6-phosphate and lysine. When incubated with fructose 6-phosphate and elevated concentrations of lysine, GfrE catalysed the formation of a compound identified as 2-epsilon-lysino-2-deoxy-6-phospho-glucose (glucoselysine 6-phosphate) by NMR. GfrE also catalysed the reciprocal conversion, i.e. the formation of fructose 6-phosphate (but not glucose 6-phosphate) from glucoselysine 6-phosphate. The equilibrium constant of this reaction (0.8 M) suggests that the enzyme serves to degrade glucoselysine 6-phosphate. In conclusion, GfrF and GfrE serve to metabolize glycation products formed from lysine and glucose (fructoselysine) or fructose (glucoselysine), via their 6-phospho derivatives. The latter are presumably formed by the putative phosphotransferase system encoded by gfrA-gfrD. The designation gfr (glycation and fructation product degradation) is proposed for this operon. This is the first description of an enzyme participating in the metabolism of fructation products. PMID:16153181

  5. 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

  6. Bioproduction of D-Tagatose from D-Galactose Using Phosphoglucose Isomerase from Pseudomonas aeruginosa PAO1.

    PubMed

    Patel, Manisha J; Patel, Arti T; Akhani, Rekha; Dedania, Samir; Patel, Darshan H

    2016-07-01

    Pseudomonas aeruginosa PAO1 phosphoglucose isomerase was purified as an active soluble form by a single-step purification using Ni-NTA chromatography that showed homogeneity on SDS-PAGE with molecular mass ∼62 kDa. The optimum temperature and pH for the maximum isomerization activity with D-galactose were 60 °C and 7.0, respectively. Generally, sugar phosphate isomerases show metal-independent activity but PA-PGI exhibited metal-dependent isomerization activity with aldosugars and optimally catalyzed the D-galactose isomerization in the presence of 1.0 mM MnCl2. The apparent Km and Vmax for D-galactose under standardized conditions were calculated to be 1029 mM (±31.30 with S.E.) and 5.95 U/mg (±0.9 with S.E.), respectively. Equilibrium reached after 180 min with production of 567.51 μM D-tagatose from 1000 mM of D-galactose. Though, the bioconversion ratio is low but it can be increased by immobilization and enzyme engineering. Although various L-arabinose isomerases have been characterized for bioproduction of D-tagatose, P. aeruginosa glucose phosphate isomerase is distinguished from the other L-arabinose isomerases by its optimal temperature (60 °C) for D-tagatose production being mesophilic bacteria, making it an alternate choice for bulk production. PMID:26922727

  7. Dosage Compensation in DROSOPHILA MELANOGASTER Triploids. II. Glucose-6-Phosphate Dehydrogenase Activity

    PubMed Central

    Maroni, Gustavo; Plaut, Walter

    1973-01-01

    The level of activity of the enzyme glucose-6-phosphate dehydrogenase was determinel in flies having seven different chromosomic constitutions. All those having an integral number of chromosomes [XAA, XXAA, XAAA, XXAAA, and XXXAAA (X=X chromosome, A=set of autosomes)] were found to have similar units of enzyme activity/mg live weight, while diploid females with a duplication and triploid females with a deficiency showed dosage effect. The amount of enzyme activity per cell, on the other hand, is also independent of the number of X's present but appears roughly proportional to the number of sets of autosomes.—It is proposed that dosage-compensated sex-linked genes are controlled by a positively acting regulatory factor(s) of autosomal origin. With this hypothesis it is possible to explain dosage compensation as a consequence of general regulatory mechanisms without invoking a special device which applies only to the X chromosomes. PMID:17248620

  8. 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

  9. 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

  10. 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.

  11. 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

  12. 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

  13. 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.

  14. Reduced methylation of PFKFB3 in cancer cells shunts glucose towards the pentose phosphate pathway

    PubMed Central

    Yamamoto, Takehiro; Takano, Naoharu; Ishiwata, Kyoko; Ohmura, Mitsuyo; Nagahata, Yoshiko; Matsuura, Tomomi; Kamata, Aki; Sakamoto, Kyoko; Nakanishi, Tsuyoshi; Kubo, Akiko; Hishiki, Takako; Suematsu, Makoto

    2014-01-01

    Haem oxygenase (HO)-1/carbon monoxide (CO) protects cancer cells from oxidative stress, but the gas-responsive signalling mechanisms remain unknown. Here we show using metabolomics that CO-sensitive methylation of PFKFB3, an enzyme producing fructose 2,6-bisphosphate (F-2,6-BP), serves as a switch to activate phosphofructokinase-1, a rate-limiting glycolytic enzyme. In human leukaemia U937 cells, PFKFB3 is asymmetrically di-methylated at R131 and R134 through modification by protein arginine methyltransferase 1. HO-1 induction or CO results in reduced methylation of PFKFB3 in varied cancer cells to suppress F-2,6-BP, shifting glucose utilization from glycolysis toward the pentose phosphate pathway. Loss of PFKFB3 methylation depends on the inhibitory effects of CO on haem-containing cystathionine β-synthase (CBS). CBS modulates remethylation metabolism, and increases NADPH to supply reduced glutathione, protecting cells from oxidative stress and anti-cancer reagents. Once the methylation of PFKFB3 is reduced, the protein undergoes polyubiquitination and is degraded in the proteasome. These results suggest that the CO/CBS-dependent regulation of PFKFB3 methylation determines directional glucose utilization to ensure resistance against oxidative stress for cancer cell survival. PMID:24633012

  15. Phosphoryl transfer from α-d-glucose 1-phosphate catalyzed by Escherichia coli sugar-phosphate phosphatases of two protein superfamily types.

    PubMed

    Wildberger, Patricia; Pfeiffer, Martin; Brecker, Lothar; Rechberger, Gerald N; Birner-Gruenberger, Ruth; Nidetzky, Bernd

    2015-03-01

    The Cori ester α-d-glucose 1-phosphate (αGlc 1-P) is a high-energy intermediate of cellular carbohydrate metabolism. Its glycosidic phosphomonoester moiety primes αGlc 1-P for flexible exploitation in glucosyl and phosphoryl transfer reactions. Two structurally and mechanistically distinct sugar-phosphate phosphatases from Escherichia coli were characterized in this study for utilization of αGlc 1-P as a phosphoryl donor substrate. The agp gene encodes a periplasmic αGlc 1-P phosphatase (Agp) belonging to the histidine acid phosphatase family. Had13 is from the haloacid dehydrogenase-like phosphatase family. Cytoplasmic expression of Agp (in E. coli Origami B) gave a functional enzyme preparation (kcat for phosphoryl transfer from αGlc 1-P to water, 40 s(-1)) that was shown by mass spectrometry to exhibit no free cysteines and the native intramolecular disulfide bond between Cys(189) and Cys(195). Enzymatic phosphoryl transfer from αGlc 1-P to water in H2 (18)O solvent proceeded with complete (18)O label incorporation into the phosphate released, consistent with catalytic reaction through O-1-P, but not C-1-O, bond cleavage. Hydrolase activity of both enzymes was not restricted to a glycosidic phosphomonoester substrate, and d-glucose 6-phosphate was converted with a kcat similar to that of αGlc 1-P. By examining phosphoryl transfer from αGlc 1-P to an acceptor substrate other than water (d-fructose or d-glucose), we discovered that Agp exhibited pronounced synthetic activity, unlike Had13, which utilized αGlc 1-P mainly for phosphoryl transfer to water. By applying d-fructose in 10-fold molar excess over αGlc 1-P (20 mM), enzymatic conversion furnished d-fructose 1-phosphate as the main product in a 55% overall yield. Agp is a promising biocatalyst for use in transphosphorylation from αGlc 1-P. PMID:25527541

  16. Acclimation of metabolism to light in A rabidopsis thaliana: the glucose 6‐phosphate/phosphate translocator GPT2 directs metabolic acclimation

    PubMed Central

    DYSON, BETH C.; ALLWOOD, J. WILLIAM; FEIL, REGINA; XU, YUN; MILLER, MATTHEW; BOWSHER, CAROLINE G.; GOODACRE, ROYSTON; LUNN, JOHN E.

    2015-01-01

    Abstract Mature leaves of plants transferred from low to high light typically increase their photosynthetic capacity. In A rabidopsis thaliana, this dynamic acclimation requires expression of GPT2, a glucose 6‐phosphate/phosphate translocator. Here, we examine the impact of GPT2 on leaf metabolism and photosynthesis. Plants of wild type and of a GPT2 knockout (gpt2.2) grown under low light achieved the same photosynthetic rate despite having different metabolic and transcriptomic strategies. Immediately upon transfer to high light, gpt2.2 plants showed a higher rate of photosynthesis than wild‐type plants (35%); however, over subsequent days, wild‐type plants acclimated photosynthetic capacity, increasing the photosynthesis rate by 100% after 7 d. Wild‐type plants accumulated more starch than gpt2.2 plants throughout acclimation. We suggest that GPT2 activity results in the net import of glucose 6‐phosphate from cytosol to chloroplast, increasing starch synthesis. There was clear acclimation of metabolism, with short‐term changes typically being reversed as plants acclimated. Distinct responses to light were observed in wild‐type and gpt2.2 leaves. Significantly higher levels of sugar phosphates were observed in gpt2.2. We suggest that GPT2 alters the distribution of metabolites between compartments and that this plays an essential role in allowing the cell to interpret environmental signals. PMID:25474495

  17. 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

  18. Combined fluxomics and transcriptomics analysis of glucose catabolism via a partially cyclic pentose phosphate pathway in Gluconobacter oxydans 621H.

    PubMed

    Hanke, Tanja; Nöh, Katharina; Noack, Stephan; Polen, Tino; Bringer, Stephanie; Sahm, Hermann; Wiechert, Wolfgang; Bott, Michael

    2013-04-01

    In this study, the distribution and regulation of periplasmic and cytoplasmic carbon fluxes in Gluconobacter oxydans 621H with glucose were studied by (13)C-based metabolic flux analysis ((13)C-MFA) in combination with transcriptomics and enzyme assays. For (13)C-MFA, cells were cultivated with specifically (13)C-labeled glucose, and intracellular metabolites were analyzed for their labeling pattern by liquid chromatography-mass spectrometry (LC-MS). In growth phase I, 90% of the glucose was oxidized periplasmically to gluconate and partially further oxidized to 2-ketogluconate. Of the glucose taken up by the cells, 9% was phosphorylated to glucose 6-phosphate, whereas 91% was oxidized by cytoplasmic glucose dehydrogenase to gluconate. Additional gluconate was taken up into the cells by transport. Of the cytoplasmic gluconate, 70% was oxidized to 5-ketogluconate and 30% was phosphorylated to 6-phosphogluconate. In growth phase II, 87% of gluconate was oxidized to 2-ketogluconate in the periplasm and 13% was taken up by the cells and almost completely converted to 6-phosphogluconate. Since G. oxydans lacks phosphofructokinase, glucose 6-phosphate can be metabolized only via the oxidative pentose phosphate pathway (PPP) or the Entner-Doudoroff pathway (EDP). (13)C-MFA showed that 6-phosphogluconate is catabolized primarily via the oxidative PPP in both phases I and II (62% and 93%) and demonstrated a cyclic carbon flux through the oxidative PPP. The transcriptome comparison revealed an increased expression of PPP genes in growth phase II, which was supported by enzyme activity measurements and correlated with the increased PPP flux in phase II. Moreover, genes possibly related to a general stress response displayed increased expression in growth phase II. PMID:23377928

  19. An alpha-glucose-1-phosphate phosphodiesterase is present in rat liver cytosol

    SciTech Connect

    Srisomsap, C.; Richardson, K.L.; Jay, J.C.; Marchase, R.B. )

    1989-12-05

    UDP-glucose:glycoprotein glucose-1-phosphotransferase (Glc-phosphotransferase) catalyzes the transfer of alpha-Glc-1-P from UDP-Glc to mannose residues on acceptor glycoproteins. The predominant acceptor for this transfer in both mammalian cells and Paramecium is a cytoplasmic glycoprotein of 62-63 kDa. When cytoplasmic proteins from rat liver were fractionated by preparative isoelectric focusing following incubation of a liver homogenate with the 35S-labeled phosphorothioate analogue of UDP-Glc ((beta-35S)UDP-Glc), the acceptor was found to have a pI of about 6.0. This fraction, when not labeled prior to the focusing, became very heavily labeled when mixed with (beta-35S). UDP-Glc and intact liver microsomes, a rich source of the Glc-phosphotransferase. In addition, it was observed that the isoelectric fractions of the cytosol having pI values of 2-3.2 contained a degradative activity, alpha-Glc-1-P phosphodiesterase, that was capable of removing alpha-Glc-1-P, monitored through radioactive labeling both in the sugar and the phosphate, as an intact unit from the 62-kDa acceptor. Identification of the product of this cleavage was substantiated by its partial transformation to UDP-Glc in the presence of UTP and UDP-Glc pyrophosphorylase. The alpha-Glc-1-P phosphodiesterase had a pH optimum of 7.5 and was not effectively inhibited by any of the potential biochemical inhibitors that were tested. Specificity for the Glc-alpha-1-P-6-Man diester was suggested by the diesterase's inability to degrade UDP-Glc or glucosylphosphoryldolichol. This enzyme may be important in the regulation of secretion since the alpha-Glc-1-P present on the 62-kDa phosphoglycoprotein appears to be removed and then rapidly replaced in response to secretagogue.

  20. Glucose-6-phosphate transport activity in liver microsomes exposed to stilbene disulfonate derivatives

    SciTech Connect

    Countaway, J.L.; Arion, W.J.

    1986-05-01

    Glucose-6-P (G6P) hydrolysis by hepatic microsomes (MS) is mediated by a coupled system composed of the G6P transporter (T1), the enzyme (E) and a phosphate transporter (T2). Zoccoli et al. concluded that T1 is a 54 kDa protein based on a linear correlation of labeling by /sup 3/H-4,4'diisothiocyano-1,2-diphenylethane-2,2'-disulfonate (/sup 3/H-H/sub 2/DIDS) and inhibition of system activity. The authors cannot support this conclusion: (1) in their hands the reaction of /sup 3/H-H/sub 2/DIDS with MS proteins is extremely nonspecific, and (2) the linear correlation must be between labeling and inhibition of T1 activity, because transport per se is not the absolute rate limiting step in hydrolysis by the system. Point 2 is readily demonstrated by examining the influence of the enzyme inhibitor, D-glucose, on the sensitivity of the system to inhibition by H/sub 2/DIDS. Studies of H/sub 2/DIDS inhibition of the system in MS from fasted and diabetic rats revealed that the observed inhibition constant for the system, K/sub i(S)/, is inversely proportional to the fraction of latent G6Pase activity (LF) seen before exposure to H/sub 2/DIDS, and K/sub i(S)/ x LF - K/sub i(T1)/, the inhibition constant for T1 activity. This relationship is derived from the equation 1/V/sub (S)/ - 1/V/sub (E)/ = 1/V/sub (T1)/, where V denotes the initial rates of S, E and T1, respectively. The latter equation can be used to calculate V/sub (T1)/ for any preparation of intact MS, and it predicts that labeling and inhibition of T1 will be linearly correlated with V/sub (T1)/ but not V/sub (S)/.

  1. Underestimation of the pentose–phosphate pathway in intact primary neurons as revealed by metabolic flux analysis

    PubMed Central

    Rodriguez-Rodriguez, Patricia; Fernandez, Emilio; Bolaños, Juan P

    2013-01-01

    The rates of glucose oxidized at glycolysis and pentose–phosphate pathway (PPP) in neurons are controversial. Using [3-3H]-, [1-14C]-, and [6-14C]glucose to estimate fluxes through these pathways in resting, intact rat cortical primary neurons, we found that the rate of glucose oxidized through PPP was, apparently, ∼14% of total glucose metabolized. However, inhibition of PPP rate-limiting step, glucose-6-phosphate (G6P) dehydrogenase, increased approximately twofold the glycolytic rate; and, knockdown of phosphoglucose isomerase increased ∼1.8-fold the PPP rate. Thus, in neurons, a considerable fraction of fructose-6-phosphate returning from the PPP contributes to the G6P pool that re-enters PPP, largely underestimating its flux. PMID:24064491

  2. 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

  3. Characterization of Morphine-Glucose-6-Phosphate Dehydrogenase Conjugates by Mass Spectrometry

    PubMed Central

    Chiu, May L.; Ytterberg, A. Jimmy; Ogorzalek Loo, Rachel R.; Loo, Joseph A.; Monbouquette, Harold G.

    2011-01-01

    A key characteristic of the analyte-reporter enzyme conjugate used in the enzyme-multiplied immunoassay technique (EMIT) is the inhibition of the conjugate enzyme upon anti-analyte antibody binding. Toward understanding the antibody-induced inhibition mechanism, characterization of morphine-glucose-6-phosphate dehydrogenase (G6PDH) conjugates as model EMIT analyte-reporter enzyme conjugates was pursued. Morphine-G6PDH conjugates were prepared by acylating predominantly the primary amines on G6PDH with morphine-3-glucuronide NHS-ester molecules. In this study, morphine-G6PDH conjugates were characterized using a combination of methods including tryptic digestion, immunoprecipitation, matrix-assisted laser desorption/ionization mass spectrometry, and electrospray ionization tandem mass spectrometry. Twenty-six conjugation sites were identified. The identified sites all were found to be primary amines. The degree of conjugation was determined to be less than the number of conjugation sites, suggesting heterogeneity within the morphine-G6PDH conjugate population. Two catalytically important residues in the active site (K22 and K183) were among the identified conjugation sites, explaining at least partially, the cause of activity loss due to the coupling reaction. PMID:21678975

  4. 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

  5. [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

  6. 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.

  7. 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

  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. Metformin-Induced Hemolytic Anemia in a Patient With Glucose-6-Phosphate Dehydrogenase Deficiency.

    PubMed

    Ruggiero, Nicole A; Kish, Troy D; Lee, Mikyung L

    2016-01-01

    Metformin, an oral antidiabetic agent, is considered the preferred first-line therapy for patients with type II diabetes. Between 2010 and 2012, it has been estimated that 14 million Americans were administered an oral antidiabetic agent, suggesting the extensive use of metformin among the diabetic population. There have been few case reports implicating metformin in causing hemolytic anemia. We present a case of a 53-year-old white male who developed hemolytic anemia after the initiation of treatment with metformin 500 mg twice daily. The patient experienced a 1.5 g/dL decrease in hemoglobin from baseline and a 2.8 mg/dL increase in total bilirubin within 1 day of treatment. Laboratory results confirmed that the patient was also glucose-6-phosphate dehydrogenase deficient. The hemolytic anemia resolved on discontinuation of metformin. Although this adverse effect seems to be rare, it is important to consider its seriousness. Clinicians should be advised to closely monitor patients newly started on metformin. PMID:25756470

  10. 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

  11. Humanized mouse model of glucose 6-phosphate dehydrogenase deficiency for in vivo assessment of hemolytic toxicity

    PubMed Central

    Rochford, Rosemary; Ohrt, Colin; Baresel, Paul C.; Campo, Brice; Sampath, Aruna; Magill, Alan J.; Tekwani, Babu L.; Walker, Larry A.

    2013-01-01

    Individuals with glucose 6-phosphate dehydrogenase (G6PD) deficiency are at risk for the development of hemolytic anemia when given 8-aminoquinolines (8-AQs), an important class of antimalarial/antiinfective therapeutics. However, there is no suitable animal model that can predict the clinical hemolytic potential of drugs. We developed and validated a human (hu)RBC-SCID mouse model by giving nonobese diabetic/SCID mice daily transfusions of huRBCs from G6PD-deficient donors. Treatment of SCID mice engrafted with G6PD-deficient huRBCs with primaquine, an 8-AQ, resulted in a dose-dependent selective loss of huRBCs. To validate the specificity of this model, we tested known nonhemolytic antimalarial drugs: mefloquine, chloroquine, doxycycline, and pyrimethamine. No significant loss of G6PD-deficient huRBCs was observed. Treatment with drugs known to cause hemolytic toxicity (pamaquine, sitamaquine, tafenoquine, and dapsone) resulted in loss of G6PD-deficient huRBCs comparable to primaquine. This mouse model provides an important tool to test drugs for their potential to cause hemolytic toxicity in G6PD-deficient populations. PMID:24101478

  12. Phosphoryl Transfer from α-d-Glucose 1-Phosphate Catalyzed by Escherichia coli Sugar-Phosphate Phosphatases of Two Protein Superfamily Types

    PubMed Central

    Wildberger, Patricia; Pfeiffer, Martin; Brecker, Lothar; Rechberger, Gerald N.; Birner-Gruenberger, Ruth

    2014-01-01

    The Cori ester α-d-glucose 1-phosphate (αGlc 1-P) is a high-energy intermediate of cellular carbohydrate metabolism. Its glycosidic phosphomonoester moiety primes αGlc 1-P for flexible exploitation in glucosyl and phosphoryl transfer reactions. Two structurally and mechanistically distinct sugar-phosphate phosphatases from Escherichia coli were characterized in this study for utilization of αGlc 1-P as a phosphoryl donor substrate. The agp gene encodes a periplasmic αGlc 1-P phosphatase (Agp) belonging to the histidine acid phosphatase family. Had13 is from the haloacid dehydrogenase-like phosphatase family. Cytoplasmic expression of Agp (in E. coli Origami B) gave a functional enzyme preparation (kcat for phosphoryl transfer from αGlc 1-P to water, 40 s−1) that was shown by mass spectrometry to exhibit no free cysteines and the native intramolecular disulfide bond between Cys189 and Cys195. Enzymatic phosphoryl transfer from αGlc 1-P to water in H218O solvent proceeded with complete 18O label incorporation into the phosphate released, consistent with catalytic reaction through O-1–P, but not C-1–O, bond cleavage. Hydrolase activity of both enzymes was not restricted to a glycosidic phosphomonoester substrate, and d-glucose 6-phosphate was converted with a kcat similar to that of αGlc 1-P. By examining phosphoryl transfer from αGlc 1-P to an acceptor substrate other than water (d-fructose or d-glucose), we discovered that Agp exhibited pronounced synthetic activity, unlike Had13, which utilized αGlc 1-P mainly for phosphoryl transfer to water. By applying d-fructose in 10-fold molar excess over αGlc 1-P (20 mM), enzymatic conversion furnished d-fructose 1-phosphate as the main product in a 55% overall yield. Agp is a promising biocatalyst for use in transphosphorylation from αGlc 1-P. PMID:25527541

  13. Discovery of a novel glucose metabolism in cancer: The role of endoplasmic reticulum beyond glycolysis and pentose phosphate shunt

    PubMed Central

    Marini, Cecilia; Ravera, Silvia; Buschiazzo, Ambra; Bianchi, Giovanna; Orengo, Anna Maria; Bruno, Silvia; Bottoni, Gianluca; Emionite, Laura; Pastorino, Fabio; Monteverde, Elena; Garaboldi, Lucia; Martella, Roberto; Salani, Barbara; Maggi, Davide; Ponzoni, Mirco; Fais, Franco; Raffaghello, Lizzia; Sambuceti, Gianmario

    2016-01-01

    Cancer metabolism is characterized by an accelerated glycolytic rate facing reduced activity of oxidative phosphorylation. This “Warburg effect” represents a standard to diagnose and monitor tumor aggressiveness with 18F-fluorodeoxyglucose whose uptake is currently regarded as an accurate index of total glucose consumption. Studying cancer metabolic response to respiratory chain inhibition by metformin, we repeatedly observed a reduction of tracer uptake facing a marked increase in glucose consumption. This puzzling discordance brought us to discover that 18F-fluorodeoxyglucose preferentially accumulates within endoplasmic reticulum by exploiting the catalytic function of hexose-6-phosphate-dehydrogenase. Silencing enzyme expression and activity decreased both tracer uptake and glucose consumption, caused severe energy depletion and decreased NADPH content without altering mitochondrial function. These data document the existence of an unknown glucose metabolism triggered by hexose-6-phosphate-dehydrogenase within endoplasmic reticulum of cancer cells. Besides its basic relevance, this finding can improve clinical cancer diagnosis and might represent potential target for therapy. PMID:27121192

  14. Discovery of a novel glucose metabolism in cancer: The role of endoplasmic reticulum beyond glycolysis and pentose phosphate shunt.

    PubMed

    Marini, Cecilia; Ravera, Silvia; Buschiazzo, Ambra; Bianchi, Giovanna; Orengo, Anna Maria; Bruno, Silvia; Bottoni, Gianluca; Emionite, Laura; Pastorino, Fabio; Monteverde, Elena; Garaboldi, Lucia; Martella, Roberto; Salani, Barbara; Maggi, Davide; Ponzoni, Mirco; Fais, Franco; Raffaghello, Lizzia; Sambuceti, Gianmario

    2016-01-01

    Cancer metabolism is characterized by an accelerated glycolytic rate facing reduced activity of oxidative phosphorylation. This "Warburg effect" represents a standard to diagnose and monitor tumor aggressiveness with (18)F-fluorodeoxyglucose whose uptake is currently regarded as an accurate index of total glucose consumption. Studying cancer metabolic response to respiratory chain inhibition by metformin, we repeatedly observed a reduction of tracer uptake facing a marked increase in glucose consumption. This puzzling discordance brought us to discover that (18)F-fluorodeoxyglucose preferentially accumulates within endoplasmic reticulum by exploiting the catalytic function of hexose-6-phosphate-dehydrogenase. Silencing enzyme expression and activity decreased both tracer uptake and glucose consumption, caused severe energy depletion and decreased NADPH content without altering mitochondrial function. These data document the existence of an unknown glucose metabolism triggered by hexose-6-phosphate-dehydrogenase within endoplasmic reticulum of cancer cells. Besides its basic relevance, this finding can improve clinical cancer diagnosis and might represent potential target for therapy. PMID:27121192

  15. Glucose 6-phosphate dehydrogenase deficient subjects may be better "storers" than donors of red blood cells.

    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

    2016-07-01

    Storage of packed red blood cells (RBCs) is associated with progressive accumulation of lesions, mostly triggered by energy and oxidative stresses, which potentially compromise the effectiveness of the transfusion therapy. Concerns arise as to whether glucose 6-phosphate dehydrogenase deficient subjects (G6PD(-)), ~5% of the population in the Mediterranean area, should be accepted as routine donors in the light of the increased oxidative stress their RBCs suffer from. To address this question, we first performed morphology (scanning electron microscopy), physiology and omics (proteomics and metabolomics) analyses on stored RBCs from healthy or G6PD(-) donors. We then used an in vitro model of transfusion to simulate transfusion outcomes involving G6PD(-) donors or recipients, by reconstituting G6PD(-) stored or fresh blood with fresh or stored blood from healthy volunteers, respectively, at body temperature. We found that G6PD(-) cells store well in relation to energy, calcium and morphology related parameters, though at the expenses of a compromised anti-oxidant system. Additional stimuli, mimicking post-transfusion conditions (37°C, reconstitution with fresh healthy blood, incubation with oxidants) promoted hemolysis and oxidative lesions in stored G6PD(-) cells in comparison to controls. On the other hand, stored healthy RBC units showed better oxidative parameters and lower removal signaling when reconstituted with G6PD(-) fresh blood compared to control. Although the measured parameters of stored RBCs from the G6PD deficient donors appeared to be acceptable, the results from the in vitro model of transfusion suggest that G6PD(-) RBCs could be more susceptible to hemolysis and oxidative stresses post-transfusion. On the other hand, their chronic exposure to oxidative stress might make them good recipients, as they better tolerate exposure to oxidatively damaged long stored healthy RBCs. PMID:27094493

  16. Glucose-6-phosphate dehydrogenase in small intestine of rabbit: biochemical properties and subcellular localization.

    PubMed

    Ninfali, P; Malatesta, M; Biagiotti, E; Aluigi, G; Gazzanelli, G

    2001-07-01

    Biochemical properties and cellular and subcellular distribution patterns of glucose-6-phosphate dehydrogenase (G6PD) were investigated in small intestine of rabbits. The specific activity of G6PD in fresh homogenates of small intestine was 19 +/- 9 IU/g protein. This value did not change significantly after dialysis. The kinetic and electrophoretic properties of the partially purified enzyme were similar to those found in other rabbit tissues. Enzyme histochemical analysis of G6PD activity using the tetrazolium salt method showed high activity in epithelial cells of villi and crypts of Lieberkuhn. The activity in acinar cells of Brunner's glands was lower than that in epithelium, whereas cells of the muscularis externa showed a very low activity. Immunohistochemical analysis showed that the amounts of G6PD protein were lower in the epithelium than in Brunner's glands and muscularis externa. The differences between distribution patterns of activity and protein of G6PD may reflect the presence of inactive enzyme molecules in Brunner's glands and muscularis externa or posttranslational activation of G6PD in epithelium. Electron microscopic immunocytochemical analysis performed with gold-labelled antibodies showed the presence of G6PD protein throughout the cytoplasm and at smooth endoplasmic reticulum in enterocytes. In Paneth cells and cells of Brunner's glands, G6PD was found in the cytoplasm, at rough endoplasmic reticulum and Golgi complex. Immunolabelling was not found in mitochondria or nuclei. Our findings show that G6PD is heterogeneously distributed in cells of the small intestine and that the enzyme is associated with rough and smooth endoplasmic reticulum to support synthetic functions in these compartments by NADPH production. PMID:11482375

  17. Evaluation of the blue formazan spot test for screening glucose 6 phosphate dehydrogenase deficiency.

    PubMed

    Pujades, A; Lewis, M; Salvati, A M; Miwa, S; Fujii, H; Zarza, R; Alvarez, R; Rull, E; Corrons, J L

    1999-06-01

    Several screening tests for glucose 6 phosphate dehydrogenase (G6PD) deficiency have been reported thus far, and a standardized method of testing was proposed by the International Council for Standardization in Hematology (ICSH). The screening test used in any particular laboratory depends upon a number of factors such as cost, time required, temperature, humidity, and availability of reagents. In this study, a direct comparison between three different G6PD screening methods has been undertaken. In 71 cases (50 hematologically normal volunteers, 9 hemizygous G6PD-deficient males, and 12 heterozygous deficient females), the blue formazan spot test (BFST) was compared with the conventional methemoglobin reduction test (HiRT) and the ICSH-recommended fluorescent spot test (FST-ICSH). In all cases, the results obtained with the three screening tests were correlated with the enzyme activity assayed spectrophotometrically. In hemizygous G6PD-deficient males, all cases were equally detected with the three methods: BFST (4.7-6.64, controls: 11.1-13.4), BMRT (score +3 in all 9 cases), and FST (no fluorescence in 9 cases). In heterozygous G6PD-deficient females, two methods detected 7 out of 12 cases (BFST: 8.71-11.75, controls: 11.1-13.4; and BMRT: score +3 in 7 cases), whereas the FST-ICSH missed all 12 cases that presented a variable degree of fluorescence. Although the sensitivity for G6PD-deficient carrier detection is the same for the BMRT and the BFST, the latter has the advantage of being semiquantitative and not merely qualitative. Unfortunately, none of the three screening tests compared here allowed the detection of the 100% heterozygote carrier state of G6PD deficiency. PMID:10407579

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

    DOE PAGESBeta

    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

  19. 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

  20. Antimalarial NADPH-Consuming Redox-Cyclers As Superior Glucose-6-Phosphate Dehydrogenase Deficiency Copycats

    PubMed Central

    Bielitza, Max; Belorgey, Didier; Ehrhardt, Katharina; Johann, Laure; Lanfranchi, Don Antoine; Gallo, Valentina; Schwarzer, Evelin; Mohring, Franziska; Jortzik, Esther; Williams, David L.; Becker, Katja; Arese, Paolo; Elhabiri, Mourad

    2015-01-01

    Abstract Aims: Early phagocytosis of glucose-6-phosphate dehydrogenase (G6PD)-deficient erythrocytes parasitized by Plasmodium falciparum were shown to protect G6PD-deficient populations from severe malaria. Here, we investigated the mechanism of a novel antimalarial series, namely 3-[substituted-benzyl]-menadiones, to understand whether these NADPH-consuming redox-cyclers, which induce oxidative stress, mimic the natural protection of G6PD deficiency. Results: We demonstrated that the key benzoylmenadione metabolite of the lead compound acts as an efficient redox-cycler in NADPH-dependent methaemoglobin reduction, leading to the continuous formation of reactive oxygen species, ferrylhaemoglobin, and subsequent haemichrome precipitation. Structure–activity relationships evidenced that both drug metabolites and haemoglobin catabolites contribute to potentiate drug effects and inhibit parasite development. Disruption of redox homeostasis by the lead benzylmenadione was specifically induced in Plasmodium falciparum parasitized erythrocytes and not in non-infected cells, and was visualized via changes in the glutathione redox potential of living parasite cytosols. Furthermore, the redox-cycler shows additive and synergistic effects in combination with compounds affecting the NADPH flux in vivo. Innovation: The lead benzylmenadione 1c is the first example of a novel redox-active agent that mimics the behavior of a falciparum parasite developing inside a G6PD-deficient red blood cell (RBC) giving rise to malaria protection, and it exerts specific additive effects that are inhibitory to parasite development, without harm for non-infected G6PD-sufficient or -deficient RBCs. Conclusion: This strategy offers an innovative perspective for the development of future antimalarial drugs for G6PD-sufficient and -deficient populations. Antioxid. Redox Signal. 22, 1337–1351. PMID:25714942

  1. 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

  2. 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.

  3. Glucose-1-Phosphate Transport into Protoplasts and Chloroplasts from Leaves of Arabidopsis1

    PubMed Central

    Fettke, Joerg; Malinova, Irina; Albrecht, Tanja; Hejazi, Mahdi; Steup, Martin

    2011-01-01

    Almost all glucosyl transfer reactions rely on glucose-1-phosphate (Glc-1-P) that either immediately acts as glucosyl donor or as substrate for the synthesis of the more widely used Glc dinucleotides, ADPglucose or UDPglucose. In this communication, we have analyzed two Glc-1-P-related processes: the carbon flux from externally supplied Glc-1-P to starch by either mesophyll protoplasts or intact chloroplasts from Arabidopsis (Arabidopsis thaliana). When intact protoplasts or chloroplasts are incubated with [U-14C]Glc-1-P, starch is rapidly labeled. Incorporation into starch is unaffected by the addition of unlabeled Glc-6-P or Glc, indicating a selective flux from Glc-1-P to starch. However, illuminated protoplasts incorporate less 14C into starch when unlabeled bicarbonate is supplied in addition to the 14C-labeled Glc-1-P. Mesophyll protoplasts incubated with [U-14C]Glc-1-P incorporate 14C into the plastidial pool of adenosine diphosphoglucose. Protoplasts prepared from leaves of mutants of Arabidopsis that lack either the plastidial phosphorylase or the phosphoglucomutase isozyme incorporate 14C derived from external Glc-1-P into starch, but incorporation into starch is insignificant when protoplasts from a mutant possessing a highly reduced ADPglucose pyrophosphorylase activity are studied. Thus, the path of assimilatory starch biosynthesis initiated by extraplastidial Glc-1-P leads to the plastidial pool of adenosine diphosphoglucose, and at this intermediate it is fused with the Calvin cycle-driven route. Mutants lacking the plastidial phosphoglucomutase contain a small yet significant amount of transitory starch. PMID:21115809

  4. Control of Hepatic Nuclear Superoxide Production by Glucose 6-Phosphate Dehydrogenase and NADPH Oxidase-4*

    PubMed Central

    Spencer, Netanya Y.; Yan, Ziying; Boudreau, Ryan L.; Zhang, Yulong; Luo, Meihui; Li, Qiang; Tian, Xin; Shah, Ajay M.; Davisson, Robin L.; Davidson, Beverly; Banfi, Botond; Engelhardt, John F.

    2011-01-01

    Redox-regulated signal transduction is coordinated by spatially controlled production of reactive oxygen species within subcellular compartments. The nucleus has long been known to produce superoxide (O2⨪); however, the mechanisms that control this function remain largely unknown. We have characterized molecular features of a nuclear superoxide-producing system in the mouse liver. Using electron paramagnetic resonance, we investigated whether several NADPH oxidases (NOX1, 2, and 4) and known activators of NOX (Rac1, Rac2, p22phox, and p47phox) contribute to nuclear O2⨪ production in isolated hepatic nuclei. Our findings demonstrate that NOX4 most significantly contributes to hepatic nuclear O2⨪ production that utilizes NADPH as an electron donor. Although NOX4 protein immunolocalized to both nuclear membranes and intranuclear inclusions, fluorescent detection of NADPH-dependent nuclear O2⨪ predominantly localized to the perinuclear space. Interestingly, NADP+ and G6P also induced nuclear O2⨪ production, suggesting that intranuclear glucose-6-phosphate dehydrogenase (G6PD) can control NOX4 activity through nuclear NADPH production. Using G6PD mutant mice and G6PD shRNA, we confirmed that reductions in nuclear G6PD enzyme decrease the ability of hepatic nuclei to generate O2⨪ in response to NADP+ and G6P. NOX4 and G6PD protein were also observed in overlapping microdomains within the nucleus. These findings provide new insights on the metabolic pathways for substrate regulation of nuclear O2⨪ production by NOX4. PMID:21212270

  5. 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.

  6. 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 Central

    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

    2015-01-01

    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

  7. The mechanism of sugar phosphate isomerization by glucosamine 6-phosphate synthase.

    PubMed Central

    Teplyakov, A.; Obmolova, G.; Badet-Denisot, M. A.; Badet, B.

    1999-01-01

    Glucosamine 6-phosphate synthase converts fructose-6P into glucosamine-6P or glucose-6P depending on the presence or absence of glutamine. The isomerase activity is associated with a 40-kDa C-terminal domain, which has already been characterized crystallographically. Now the three-dimensional structures of the complexes with the reaction product glucose-6P and with the transition state analog 2-amino-2-deoxyglucitol-6P have been determined. Glucose-6P binds in a cyclic form whereas 2-amino-2-deoxyglucitol-6P is in an extended conformation. The information on ligand-protein interactions observed in the crystal structures together with the isotope exchange and site-directed mutagenesis data allow us to propose a mechanism of the isomerase activity of glucosamine-6P synthase. The sugar phosphate isomerization involves a ring opening step catalyzed by His504 and an enolization step with Glu488 catalyzing the hydrogen transfer from C1 to C2 of the substrate. The enediol intermediate is stabilized by a helix dipole and the epsilon-amino group of Lys603. Lys485 may play a role in deprotonating the hydroxyl O1 of the intermediate. PMID:10091662

  8. Substrate product equilibrium on a reversible enzyme, triosephosphate isomerase

    PubMed Central

    Rozovsky, Sharon; McDermott, Ann E.

    2007-01-01

    The highly efficient glycolytic enzyme, triosephosphate isomerase, is expected to differentially stabilize the proposed stable reaction species: ketone, aldehyde, and enediol(ate). The identity and steady-state populations of the chemical entities bound to triosephosphate isomerase have been probed by using solid- and solution-state NMR. The 13C-enriched ketone substrate, dihydroxyacetone phosphate, was bound to the enzyme and characterized at steady state over a range of sample conditions. The ketone substrate was observed to be the major species over a temperature range from −60°C to 15°C. Thus, there is no suggestion that the enzyme preferentially stabilizes the reactive intermediate or the product. The predominance of dihydroxyacetone phosphate on the enzyme would support a mechanism in which the initial proton abstraction in the reaction from dihydroxyacetone phosphate to d-glyceraldehyde 3-phosphate is significantly slower than the subsequent chemical steps. PMID:17287353

  9. Light modulation of glyceraldehyde-3-phosphate dehydrogenase and glucose-6-phosphate dehydrogenase by photosynthetic electron flow in pea chloroplasts

    SciTech Connect

    Akamba, L.M.; Anderson, L.E.

    1981-02-01

    Light activation of NADP-linked glyceraldehyde-3-P dehydrogenase (EC 1.2.1.13) and light inactivation of glucose-6-P dehydrogenase (EC 1.1.1.49) appear to be modulated within pea leaf chloroplasts by mediators which are reduced by photosynthetic electron flow from the photosystem I reaction center. Dichlorophenyl-1,1-dimethylurea inhibition of this modulation can be completely reversed by ascorbate plus 2,6-dichlorophenolindophenol in broken chloroplasts, but not in intact chloroplasts. Intact chloroplasts are impermeable to 2,6-dichlorophenolindophenol at pH 7.5. Studies on the effect of light in reconstituted chloroplasts with photosystem I-enriched particles in the place of whole thylakoids revealed that photosystem I participants in the light modulation of NADP-linked glyceraldehyde-3-P dehydrogenase and of glucose-6-P dehydrogenase.

  10. 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

  11. Prevalence and Molecular Characterization of Glucose-6-Phosphate Dehydrogenase Deficiency at the China-Myanmar Border

    PubMed Central

    Liu, Rong; Luo, Lan; Yang, Yuling; Zhang, Lu; Liu, Huaie; Zhang, Wen; Fan, Zhixiang; Yang, Zhaoqing; Cui, Liwang; He, Yongshu

    2015-01-01

    Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked hereditary disease that predisposes red blood cells to oxidative damage. G6PD deficiency is particularly prevalent in historically malaria-endemic areas. Use of primaquine for malaria treatment may result in severe hemolysis in G6PD deficient patients. In this study, we systematically evaluated the prevalence of G6PD deficiency in the Kachin (Jingpo) ethnic group along the China-Myanmar border and determined the underlying G6PD genotypes. We surveyed G6PD deficiency in 1770 adult individuals (671 males and 1099 females) of the Kachin ethnicity using a G6PD fluorescent spot test. The overall prevalence of G6PD deficiency in the study population was 29.6% (523/1770), among which 27.9% and 30.6% were males and females, respectively. From these G6PD deficient samples, 198 unrelated individuals (147 females and 51 males) were selected for genotyping at 11 known G6PD single nucleotide polymorphisms (SNPs) in Southeast Asia (ten in exons and one in intron 11) using a multiplex SNaPshot assay. Mutations with known association to a deficient phenotype were detected in 43.9% (87/198) of cases, intronic and synonymous mutations were detected alone in 34.8% (69/198) cases and no mutation were found in 21.2% (42/198) cases. Five non-synonymous mutations, Mahidol 487G>A, Kaiping 1388G>A, Canton 1376G>T, Chinese 4 392G>T, and Viangchan 871G>A were detected. Of the 87 cases with known deficient mutations, the Mahidol variant was the most common (89.7%; 78/87), followed by the Kaiping (8.0%; 7/87) and the Viangchan (2.2%; 2/87) variants. The Canton and Chinese 4 variants were found in 1.1% of these 87 cases. Among them, two females carried the Mahidol/Viangchan and Mahidol/Kaiping double mutations, respectively. Interestingly, the silent SNPs 1311C>T and IVS11nt93T>C both occurred in the same 95 subjects with frequencies at 56.4% and 23.5% in tested females and males, respectively (P<0.05). It is noteworthy that 24

  12. The prevalence of glucose-6-phosphate dehydrogenase deficiency in Gambian school children

    PubMed Central

    2014-01-01

    Background Primaquine, the only available drug effective against Plasmodium falciparum sexual stages, induces also a dose-dependent haemolysis, especially in glucose-6-phosphate dehydrogenase deficient (G6PDd) individuals. Therefore, it is important to determine the prevalence of this deficiency in areas that would potentially benefit from its use. The prevalence of G6PD deficiency by genotype and enzyme activity was determined in healthy school children in The Gambia. Methods Blood samples from primary school children collected during a dry season malaria survey were screened for G6PDd and malaria infection. Genotypes for allele mutations reported in the country; 376, 202A-, 968A- and 542 were analysed while enzyme activity (phenotype) was assayed using a semi-quantitative commercial test kit. Enzyme activity values were fitted in a finite mixture model to determine the distribution and calculate a cut-off for deficiency. The association between genotype and phenotype for boys and girls as well as the association between mutant genotype and deficient phenotype was analysed. Results Samples from 1,437 children; 51% boys were analysed. The prevalence of P. falciparum malaria infection was 14%. The prevalence of the 202A-, 968 and 542 mutations was 1.8%, 2.1% and 1.0%, respectively, and higher in boys than in girls. The prevalence of G6PDd phenotype was 6.4% (92/1,437), 7.8% (57/728) in boys and 4.9% (35/709) in girls with significantly higher odds in the former (OR 1.64, 95% CI 1.05, 2.53, p = 0.026). The deficient phenotype was associated with reduced odds of malaria infection (OR 0.77, 95% CI 0.36, 1.62, p = 0.49). Conclusions There is a weak association between genotype and phenotype estimates of G6PDd prevalence. The phenotype expression of deficiency represents combinations of mutant alleles rather than specific mutations. Genotype studies in individuals with a deficient phenotype would help identify alleles responsible for haemolysis. PMID:24742291

  13. Hereditary sideroblastic anemia and glucose-6-phosphate dehydrogenase deficiency in a negro family

    PubMed Central

    Prasad, Ananda S.; Tranchida, Liborio; Konno, Edward T.; Berman, Lawrence; Albert, Samuel; Sing, Charles F.; Brewer, George J.

    1968-01-01

    Detailed clinical and genetic studies have been performed in a Negro family, which segregated for sex-linked sideroblastic anemia and glucose-6-phosphate dehydrogenase (G-6-DP) deficiency. This is the first such pedigree reported. Males affected with sideroblastic anemia had growth retardation, hypochromic microcytic anemia, elevated serum iron, decreased unsaturated iron-binding capacity, increased 59Fe clearance, low 59Fe incorporation into erythrocytes, normal erythrocyte survival (51Cr), normal hemoglobin electrophoretic pattern, erythroblastic hyperplasia of marrow with increased iron, and marked increase in marrow sideroblasts, particularly ringed sideroblasts. Perinuclear deposition of ferric aggregates was demonstrated to be intramitochondrial by electron microscopy. Female carriers of the sideroblastic gene were normal but exhibited a dimorphic population of erythrocytes including normocytic and microcytic cells. The bone marrow studies in the female (mother) showed ringed marrow sideroblasts. Studies of G-6-PD involved the methemoglobin elution test for G-6-PD activity of individual erythrocytes, quantitative G-6-PD assay, and electrophoresis. In the pedigree, linkage information was obtained from a doubly heterozygous woman, four of her sons, and five of her daughters. Three sons were doubly affected, and one was normal. One daughter appeared to be a recombinant. The genes appeared to be linked in the coupling phase in the mother. The maximum likelihood estimate of the recombination value was 0.14. By means of Price-Jones curves, the microcytic red cells in peripheral blood were quantitated in female carriers. The sideroblast count in the bone marrow in the mother corresponded closely to the percentage of microcytic cells in peripheral blood. This is the second example in which the cellular expression of a sex-linked trait has been documented in the human red cells, the first one being G-6-PD deficiency. The coexistence of the two genes in doubly

  14. Mechanism of glucose-6-phosphate dehydrogenase-mediated regulation of coronary artery contractility.

    PubMed

    Ata, Hirotaka; Rawat, Dhwajbhadur K; Lincoln, Thomas; Gupte, Sachin A

    2011-06-01

    We previously identified glucose-6-phosphate dehydrogenase (G6PD) as a regulator of vascular smooth muscle contraction. In this study, we tested our hypothesis that G6PD activated by KCl via a phosphatase and tensin homologue deleted on chromosome 10 (PTEN)-protein kinase C (PKC) pathway increases vascular smooth muscle contraction and that inhibition of G6PD relaxes smooth muscle by decreasing intracellular Ca(2+) ([Ca(2+)](i)) and Ca(2+) sensitivity to the myofilament. Here we show that G6PD is activated by membrane depolarization via PKC and PTEN pathway and that G6PD inhibition decreases intracellular free calcium ([Ca(2+)](i)) in vascular smooth muscle cells and thus arterial contractility. In bovine coronary artery (CA), KCl (30 mmol/l) increased PKC activity and doubled G6PD V(max) without affecting K(m). KCl-induced PKC and G6PD activation was inhibited by bisperoxo(pyridine-2-carboxyl)oxovanadate (Bpv; 10 μmol/l), a PTEN inhibitor, which also inhibited (P < 0.05) KCl-induced CA contraction. The G6PD blockers 6-aminonicotinamide (6AN; 1 mmol/l) and epiandrosterone (EPI; 100 μmol/l) inhibited KCl-induced increases in G6PD activity, [Ca(2+)](i), Ca(2+)-dependent myosin light chain (MLC) phosphorylation, and contraction. Relaxation of precontracted CA by 6AN and EPI was not blocked by calnoxin (10 μmol/l), a plasma membrane Ca(2+) ATPase inhibitor or by lowering extracellular Na(+), which inhibits the Na(+)/Ca(2+) exchanger (NCX), but cyclopiazonic acid (200 μmol/l), a sarcoplasmic reticulum Ca(2+) ATPase inhibitor, reduced (P < 0.05) 6AN- and EPI-induced relaxation. 6AN also attenuated phosphorylation of myosin phosphatase target subunit 1 (MYPT1) at Ser855, a site phosphorylated by Rho kinase, inhibition of which reduced (P < 0.05) KCl-induced CA contraction and 6AN-induced relaxation. By contrast, 6AN increased (P < 0.05) vasodilator-stimulated phosphoprotein (VASP) phosphorylation at Ser239, indicating that inhibition of G6PD increases PKA or PKG

  15. 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

  16. Medications and glucose-6-phosphate dehydrogenase deficiency: an evidence-based review.

    PubMed

    Youngster, Ilan; Arcavi, Lidia; Schechmaster, Renata; Akayzen, Yulia; Popliski, Hen; Shimonov, Janna; Beig, Svetlana; Berkovitch, Matitiahu

    2010-09-01

    Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human enzyme defect and one of the most common genetic disorders worldwide, with an estimated 400 million people worldwide carrying a mutation in the G6PD gene that causes deficiency of the enzyme. Although drug-induced haemolysis is considered the most common adverse clinical consequence of G6PD deficiency, significant confusion exists regarding which drugs can cause haemolytic anaemia in patients with G6PD deficiency. In the absence of consensus among physicians, patients are subject to conflicting advice, causing uncertainty and distress. In the current review we aimed, by thorough search of the medical literature, to collect evidence on which to base decisions either to prohibit or allow the use of various medications in patients with G6PD deficiency. A literature search was conducted during May 2009 for studies and case reports on medication use and G6PD deficiency using the following sources: MEDLINE (1966-May 2009), PubMed (1950-May 2009), the Cochrane database of systematic reviews (2009), and major pharmacology, internal medicine, haematology and paediatric textbooks. After assessing the literature, we divided medications into one of three groups: medications that should be avoided in individuals with G6PD deficiency, medications that were considered unsafe by at least one source, but according to our review can probably be given safely in normal therapeutic dosages to individuals with G6PD deficiency as evidence does not contravene their use, and medications where no evidence at all was found to contravene their use in G6PD-deficient patients. It is reasonable to conclude that, over time, many compounds have been wrongly cited as causing haemolysis because they were administered to patients experiencing an infection-related haemolytic episode. We found solid evidence to prohibit only seven currently used medications: dapsone, methylthioninium chloride (methylene blue), nitrofurantoin

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

    PubMed

    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

  18. Effect of vitamin K1 on glucose-6-phosphate dehydrogenase deficient neonatal erythrocytes in vitro

    PubMed Central

    Kaplan, M.; Waisman, D.; Mazor, D.; Hammerman, C.; Bader, D.; Abrahamov, A.; Meyerstein, N.

    1998-01-01

    AIM—To determine whether vitamin K1, which is routinely administered to neonates, could act as an exogenous oxidising agent and be partly responsible for haemolysis in glucose-6-phosphat-dehydrogenase (G-6-PD).
METHODS—G-6-PD deficient (n=7) and control (n=10) umbilical cord blood red blood cells were incubated in vitro with a vitamin K1 preparation (Konakion). Two concentrations of Vitamin K1 were used, both higher than that of expected serum concentrations, following routine injection of 1 mg vitamin K1. Concentrations of reduced glutathione (GSH) and methaemoglobin, indicators of oxidative red blood cell damage, were determined before and after incubation, and the mean percentage change from baseline calculated.
RESULTS—Values (mean (SD)) for GSH, at baseline, and after incubation with vitamin K1 at concentrations of 44 and 444 µM, respectively, and percentage change from baseline (mean (SD)) were 1.97 + 0.31µmol/g haemoglobin, 1.89 ± 0.44 µmol/g (-4.3 ± 13.1%), and 1.69± 0.41 µmol/g (-14.5 ±9.3%) for the G-6-PD deficient red blood cells, and 2.27 ± 0.31 µmol/g haemoglobin, 2.09 ± 0.56 µmol/g (−7.2 ± 23.2%), and 2.12 ± 0.38 µmol/g (−6.0 + 14.1%) for the control cells. For methaemoglobin (percentage of total haemoglobin), the corresponding values were 2.01± 0.53%, 1.93 ± 0.37% (−0.6± 17.4%) and 2.06 ± 0.43% (5.7 ± 14.2%) for the G-6-PD deficient red blood cells, and 1.56 ± 0.74%, 1.70 ± 0.78% (12.7 ± 21.9%), and 1.78 ± 0.71% (20.6 ± 26.8%) for the control red blood cells. None of the corresponding percentage changes from baseline was significantly different when G-6-PD deficient and control red blood cells were compared.
CONCLUSIONS—These findings suggest that G-6-PD deficient red blood cells are not at increased risk of oxidative damage from vitamin K1.

 PMID:10194997

  19. 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

  20. 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

  1. The Preterm Infant: A High-Risk Situation for Neonatal Hyperbilirubinemia Due to Glucose-6-Phosphate Dehydrogenase Deficiency.

    PubMed

    Kaplan, Michael; Hammerman, Cathy; Bhutani, Vinod K

    2016-06-01

    Prematurity and glucose-6-phosphate dehydrogenase (G6PD) deficiency are risk factors for neonatal hyperbilirubinemia. The 2 conditions may interact additively or synergistically, contributing to extreme hyperbilirubinemia, with the potential for bilirubin neurotoxicity. This hyperbilirubinemia is the result of sudden, unpredictable, and acute episodes of hemolysis in combination with immaturity of bilirubin elimination, primarily of conjugation. Avoidance of contact with known triggers of hemolysis in G6PD-deficient individuals will prevent some, but not all, episodes of hemolysis. All preterm infants with G6PD deficiency should be vigilantly observed for the development of jaundice both in hospital and after discharge home. PMID:27235211

  2. 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.

  3. Cloning, biochemical characterisation, tissue localisation and possible post-translational regulatory mechanism of the cytosolic phosphoglucose isomerase from developing sunflower seeds.

    PubMed

    Troncoso-Ponce, M A; Rivoal, J; Cejudo, F J; Dorion, S; Garcés, R; Martínez-Force, E

    2010-09-01

    Lipid biosynthesis in developing sunflower (Helianthus annuus L.) seeds requires reducing power. One of the main sources of cellular NADPH is the oxidative pentose phosphate pathway (OPPP), generated from the oxidation of glucose-6-phosphate. This glycolytic intermediate, which can be imported to the plastid and enter in the OPPP, is the substrate and product of cytosolic phosphoglucose isomerase (cPGI, EC 5.3.1.9). In this report, we describe the cloning of a full-length cDNA encoding cPGI from developing sunflower seeds. The sequence was predicted to code for a protein of 566 residues characterised by the presence of two sugar isomerase domains. This cDNA was heterologously expressed in Escherichia coli as a His-tagged protein. The recombinant protein was purified using immobilised metal ion affinity chromatography and biochemically characterised. The enzyme had a specific activity of 1,436 micromol min(-1) mg(-1) and 1,011 micromol min(-1) mg(-1) protein when the reaction was initiated with glucose-6-phosphate and fructose-6-phosphate, respectively. Activity was not affected by erythrose-4-phosphate, but was inhibited by 6-P gluconate and glyceraldehyde-3-phosphate. A polyclonal immune serum was raised against the purified enzyme, allowing the study of protein levels during the period of active lipid synthesis in seeds. These results were compared with PGI activity profiles and mRNA expression levels obtained from Q-PCR studies. Our results point to the existence of a possible post-translational regulatory mechanism during seed development. Immunolocalisation of the protein in seed tissues further indicated that cPGI is highly expressed in the procambial ring. PMID:20628759

  4. Overcompensation in Response to Herbivory in Arabidopsis thaliana: The Role of Glucose-6-Phosphate Dehydrogenase and the Oxidative Pentose-Phosphate Pathway

    PubMed Central

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

    2013-01-01

    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. PMID:23934891

  5. Sphingosine-1-phosphate receptor 2 mediates endothelial cells dysfunction by PI3K-Akt pathway under high glucose condition.

    PubMed

    Liu, Weihua; Liu, Bin; Liu, Shaojun; Zhang, Jingzhi; Lin, Shuangfeng

    2016-04-01

    Endothelial dysfunction is believed the early stage of development of diabetic cardiovascular complications. Sphingosine-1-phosphate (S1P) regulates various biological activities by binding to sphingosine-1-phosphate receptors (S1PRs) including S1PR1-S1PR5. In the present study, the role of S1P receptors in S1P-induced human coronary artery endothelial cells (HCAECs) dysfunction under high glucose condition was investigated and the underlying mechanism was explored. S1PR1-S1PR5 mRNA levels were detected by quantitative Real-time PCR. NO level and polymorphonuclear neutrophils (PMN)-endothelial cells adhesion were measured by nitrate reductase and myeloperoxidase colorimetric method, respectively. Protein levels of endothelial nitric oxide synthase (eNOS), vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1(ICAM-1), phosphatidylinositol 3-kinase (PI3K) and Akt were measured by Western blot analysis. S1PR2 were found the predominant S1P receptor expressed in HCAECs exposed to high glucose. NO level and eNOS activity were remarkably decreased, while PMN adhesion, VCAM-1 and ICAM-1 protein levels were increased significantly by S1P treatment in HCAECs exposed to high glucose and normal glucose. Blockage of S1PR2 with specific antagonist JTE-013 and small interfering RNA (siRNA) resulted in enhanced NO level and eNOS activity as well as decreased PMN adhesion, reduced protein levels of VCAM-1 and ICAM-1 induced by S1P. Furthermore, Phosphor-PI3K and phosphor-Akt level were markedly increased by S1PR2 blockade in S1P-treated cells exposed to high glucose, which were suppressed by PI3K inhibitor wortmannin. In conclusion, S1P/S1PR2 mediated endothelial dysfunction partly by inhibiting PI3K/Akt signaling pathway under high glucose condition. S1PR2 blockage could ameliorate endothelial dysfunction which might provide a potential therapeutic strategy for diabetic vascular complications. PMID:26921757

  6. 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.

  7. Cloning, expression and characterization of glucokinase gene involved in the glucose-6- phosphate formation in Staphylococcus aureus

    PubMed Central

    Lakshmi, Hanumanthu Prasanna; Yeswanth, Sthanikam; Prasad, Uppu Venkateswara; Vasu, Dudipeta; Swarupa, Vimjam; Kumar, Pasupuleti Santhosh; Narasu, Mangamoori Lakshmi; Krishna Sarma, Potukuchi Venkata Gurunadha

    2013-01-01

    Glucose-6-phosphate (G-6-P) formation in Staphylococcus aureus is catalysed by glucokinase (glkA) gene under high glucose concentration leading to upregulation of various pathogenic factors; therefore the present study is aimed in the cloning and characterization of glk A gene from S. aureus ATCC12600. The glk A gene was cloned in the Sma I site of pQE 30, sequenced (Accession number: JN645812) and expressed in E. coli DH5α. The recombinant glk A expressed from the resultant glk A 1 clone was purified using nickel metal chelate chromatography, the pure enzyme gave single band in SDS-PAGE with molecular weight of 33kDa. The rglk A showed very high affinity to glucose Km 5.1±0.06mM with Hill coefficient of 1.66±0.032mM. Analysis of glucokinase sequence of S. aureus showed presence of typical ATP binding site and ROK motif CNCGRSGCIE. Sequentially and phylogenetically S. aureus glk A exhibited low identity with other bacterial glk A and 21% homology with human glucokinase (GCK). Functionally, S. aureus glk A showed higher rate of G-6-P formation compared to human GCK which may have profound role in the pathogenesis. PMID:23519063

  8. Availability of phosphate for phytoplankton and bacteria and of glucose for bacteria at different pCO2 levels in a mesocosm study

    NASA Astrophysics Data System (ADS)

    Tanaka, T.; Thingstad, T. F.; Løvdal, T.; Grossart, H.-P.; Larsen, A.; Allgaier, M.; Meyerhöfer, M.; Schulz, K. G.; Wohlers, J.; Zöllner, E.; Riebesell, U.

    2008-05-01

    Availability of phosphate for phytoplankton and bacteria and of glucose for bacteria at different pCO2 levels were studied in a mesocosm experiment (PeECE III). Using nutrient-depleted SW Norwegian fjord waters, three different levels of pCO2 (350 μatm: 1×CO2; 700 μatm: 2×CO2; 1050 μatm: 3×CO2) were set up, and nitrate and phosphate were added at the start of the experiment in order to induce a phytoplankton bloom. Despite similar responses of total particulate P concentration and phosphate turnover time at the three different pCO2 levels, the size distribution of particulate P and 33PO4 uptake suggested that phosphate transferred to the >10 μm fraction was greater in the 3×CO2 mesocosm during the first 6-10 days when phosphate concentration was high. During the period of phosphate depletion (after Day 12), specific phosphate affinity and specific alkaline phosphatase activity (APA) suggested a P-deficiency (i.e. suboptimal phosphate supply) rather than a P-limitation for the phytoplankton and bacterial community at the three different pCO2 levels. Specific phosphate affinity and specific APA tended to be higher in the 3×CO2 than in the 2×CO2 and 1×CO2 mesocosms during the phosphate depletion period, although no statistical differences were found. Glucose turnover time was correlated significantly and negatively with bacterial abundance and production but not with the bulk DOC concentration. This suggests that even though constituting a small fraction of the bulk DOC, glucose was an important component of labile DOC for bacteria. Specific glucose affinity of bacteria behaved similarly at the three different pCO2 levels with measured specific glucose affinities being consistently much lower than the theoretical maximum predicted from the diffusion-limited model. This suggests that bacterial growth was not severely limited by the glucose availability. Hence, it seems that the lower availability of inorganic nutrients after the phytoplankton bloom reduced

  9. 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. PMID:26585224

  10. Further evidence for a two-step model of glucose-transport regulation. Inositol phosphate-oligosaccharides regulate glucose-carrier activity.

    PubMed Central

    Obermaier-Kusser, B; Mühlbacher, C; Mushack, J; Seffer, E; Ermel, B; Machicao, F; Schmidt, F; Häring, H U

    1989-01-01

    The insulin effect on glucose uptake is not sufficiently explained by a simple glucose-carrier translocation model. Recent studies rather suggest a two-step model of carrier translocation and carrier activation. We used several pharmacological tools to characterize the proposed model further. We found that inositol phosphate (IP)-oligosaccharides isolated from the drug Actovegin, as well as the alkaloid vinblastine, show a partial insulin-like effect on glucose-transport activity of fat-cells (3-O-methylglucose uptake, expressed as % of equilibrium value per 4 s: basal 5.8%, insulin 59%, IP-oligosaccharides 30%, vinblastine 29%) without inducing carrier translocation. On the other hand, two newly developed anti-diabetic compounds (alpha-activated carbonic acids, BM 130795 and BM 13907) induced carrier translocation to the same extent as insulin and phorbol esters [cytochalasin-B-binding sites in plasma membranes: basal 5 pmol/mg of protein, insulin 13 pmol/mg of protein, TPA (12-O-tetradecanoylphorbol 13-acetate) 11.8 pmol/mg of protein, BM 130795 10.8 pmol/mg of protein], but produce also only 40-50% of the insulin effect on glucose-transport activity (basal 5.8%, insulin 59%, TPA 23%, BM 130795 35%). Almost the full insulin effect was mimicked by a combination of phorbol esters and IP-oligosaccharides (basal 7%, insulin 50%, IP-oligosaccharides 30%, TPA 23%, IP-oligosaccharides + TPA 45%). None of these substances stimulated insulin-receptor kinase in vitro or in vivo, suggesting a post-kinase site of action. The data confirm the following aspects of the proposed model: (1) carrier translocation and carrier activation are two independently regulated processes; (2) the full insulin effect is mimicked only by a simultaneous stimulation of carrier translocation and intrinsic carrier activity, suggesting that insulin acts through a synergism of both mechanisms; (3) IP-oligosaccharides might be involved in the transmission of a stimulatory signal on carrier activity

  11. Apoptotic effects and glucose-6-phosphate dehydrogenase responses in liver and gill tissues of rainbow trout treated with chlorpyrifos.

    PubMed

    Topal, Ahmet; Atamanalp, Muhammed; Oruç, Ertan; Kırıcı, Muammer; Kocaman, Esat Mahmut

    2014-12-01

    We investigated apoptotic effects and changes in glucose-6-phosphate dehydrogenase (G6PD) enzyme activity in liver and gill tissues of fish exposed to chlorpyrifos. Three different chlorpyrifos doses (2.25, 4.5 and 6.75 μg/L) were administrated to rainbow trout at different time intervals (24, 48, 72 and 96 h). Acute exposure to chlorpyrifos showed time dependent decrease in G6PD enzyme activity at all concentrations (p < 0.05). Immunohistochemical results showed that chlorpyrifos caused mucous cell loss in gill tissue and apoptosis via caspase-3 activation in fish. The present study suggested that chlorpyrifos inhibits G6PD enzyme and causes mucous cell loss in gill and apoptosis in gill and liver tissues. PMID:25438950

  12. 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.

  13. Glucose-6-phosphate-dehydrogenase deficiency and its correlation with other risk factors in jaundiced newborns in Southern Brazil

    PubMed Central

    Carvalho, Clarissa Gutiérrez; Castro, Simone Martins; Santin, Ana Paula; Zaleski, Carina; Carvalho, Felipe Gutiérrez; Giugliani, Roberto

    2011-01-01

    Objective To evaluate the correlation between glucose-6-phosphate-dehydrogenase (G6PD) deficiency and neonatal jaundice. Methods Prospective, observational case-control study was conducted on 490 newborns admitted to Hospital de Clínicas de Porto Alegre for phototherapy, who all experienced 35 or more weeks of gestation, from March to December 2007. Enzymatic screening of G6PD activity was performed, followed by PCR. Results There was prevalence of 4.6% and a boy-girl ratio of 3:1 in jaundiced newborns. No jaundiced neonate with ABO incompatibility presented G6PD deficiency, and no Mediterranean mutation was found. A higher proportion of deficiency was observed in Afro-descendants. There was no association with UGT1A1 variants. Conclusions G6PD deficiency is not related to severe hyperbilirubinemia and considering the high miscegenation in this area of Brazil, other gene interactions should be investigated. PMID:23569738

  14. Expression, purification, crystallization and preliminary X-ray analysis of glucose-1-phosphate uridylyltransferase (GalU) from Erwinia amylovora

    PubMed Central

    Toccafondi, Mirco; Cianci, Michele; Benini, Stefano

    2014-01-01

    Glucose-1-phosphate uridylyltransferase from Erwinia amylovora CFPB1430 was expressed as a His-tag fusion protein in Escherichia coli. After tag removal, the purified protein was crystallized from 100 mM Tris pH 8.5, 2 M ammonium sulfate, 5% ethylene glycol. Diffraction data sets were collected to a maximum resolution of 2.46 Å using synchrotron radiation. The crystals belonged to the hexagonal space group P62, with unit-cell parameters a = 80.67, b = 80.67, c = 169.18. The structure was solved by molecular replacement using the structure of the E. coli enzyme as a search model. PMID:25195902

  15. Diverse point mutations in the human glucose-6-phosphate dehydrogenase gene cause enzyme deficiency and mild or severe hemolytic anemia

    SciTech Connect

    Vulliamy, T.J.; D'Urso, M.; Battistuzzi, G.; Estrada, M.; Foulkes, N.S.; Martini, G.; Calabro, V.; Poggi, V.; Giordano, R.; Town, M.; Luzzatto, L.; Persico, M.G. )

    1988-07-01

    Glucose-6-phosphate dehydrogenase deficiency is a common genetic abnormality affecting an estimated 400 million people worldwide. Clinical and biochemical analyses have identified many variants exhibiting a range of phenotypes, which have been well characterized from the hematological point of view. However, until now, their precise molecular basis has remained unknown. The authors have cloned and sequenced seven mutant G6PD alleles. In the nondeficient polymorphic African variant G6PD A they have found a single point mutation. The other six mutants investigated were all associated with enzyme deficiency. The mutations observed show a striking predominance of C {yields} T transitions, with CG doublets involved in four of seven cases. Thus, diverse point mutations may account largely for the phenotypic heterogeneity of G6PD deficiency.

  16. Molecular cloning and nucleotide sequence of cDNA for human glucose-6-phosphate dehydrogenase variant A(-).

    PubMed Central

    Hirono, A; Beutler, E

    1988-01-01

    Glucose-6-phosphate dehydrogenase (G6PD; D-glucose-6-phosphate:NADP+ oxidoreductase, EC 1.1.1.49) A(-) is a common variant in Blacks that causes sensitivity to drug-and infection-induced hemolytic anemia. A cDNA library was constructed from Epstein-Barr virus-transformed lymphoblastoid cells from a male who was G6PD A(-). One of four cDNA clones isolated contained a sequence not found in the other clones nor in the published cDNA sequence. Consisting of 138 bases and coding 46 amino acids, this segment of cDNA apparently is derived from the alternative splicing involving the 3' end of intron 7. Comparison of the remaining sequences of these clones with the published sequence revealed three nucleotide substitutions: C33----G, G202----A, and A376----G. Each change produces a new restriction site. Genomic DNA from five G6PD A(-) individuals was amplified by the polymerase chain reaction. The base substitution at position 376, identical to the substitution that has been reported in G6PD A(+), was present in all G6PD A(-) samples and none of the control G6PD B(+) samples examined. The substitution at position 202 was found in four of the five G6PD A(-) samples and no normal control sample. At position 33 guanine was found in all G6PD A(-) samples and seven G6PD B(+) control samples and is, presumably, the usual nucleotide found at this position. The finding of the same mutation in G6PD A(-) as is found in G6PD A(+) strongly suggests that the G6PD A(-) mutation arose in an individual with G6PD A(+), adding another mutation that causes the in vivo instability of this enzyme protein. Images PMID:2836867

  17. Identification of Mutation of Glucose-6-Phosphate Dehy–drogenase (G6PD) in Iran: Meta- analysis Study

    PubMed Central

    MOOSAZADEH, Mahmood; NEKOEI-MOGHADAM, Mahmood; ALIRAM–ZANY, Maryam; AMIRESMAILI, Mohammadreza

    2013-01-01

    Abstract Background Glucose-6-phosphate dehydrogenase is one of the most common genetic deficiencies, which approximately 400 million people in the world suffer from. According to authors’ initial search, numerous studies have been carried out in Iran regarding molecular variants of this enzyme. Thus, this meta-analysis presented a reliable estimation about prevalence of different types of molecular mutations of G6PD Enzyme in Iran. Methods Keywords “glucose 6 phosphate dehydrogenase or G6PD, Mediterranean or Chatham or Cosenza and mutation, Iran or Iranian and their Persian equivalents” were searched in different databases. Moreover, reference list of the published studies were examined to increase sensitivity and to select more studies. After studying titles and abstracts of retrieved articles, excluding the repeated and unrelated ones, and evaluating quality of articles, documents were selected. Data was analyzed using STATA. Results After performing systematic review, 22 papers were entered this meta-analysis and 1698 subjects were examined concerning G6PD molecular mutation. In this meta-analysis, prevalence of Mediterranean mutation, Chatham mutation and Cosenza mutation in Iran was estimated 78.2%, 9.1% and 0.5% respectively. Conclusions This meta-analysis showed that in spite of prevalence of different types of G6PD molecular mutations in center, north, north-west and west of Iran, the most common molecular mutations in people with G6PD deficiency in Iran, like other Mediterranean countries and countries around Persian Gulf, were Mediterranean mutation, Chatham mutation and Cosenza mutation. It is also recommended that future studies may focus on races and regions which haven’t been taken into consideration up to now. PMID:26060661

  18. ISOLATION OF A PRECURSOR AND A NASCENT CHAIN FORM OF GLUCOSE-6-PHOSPHATE DEHYDROGENASE FROM RAT UTERUS AND REGULATION OF PRECURSOR PROCESSING BY ESTRADIOL

    EPA Science Inventory

    SDS-polyacrylamide gel electrophoresis of anti-glucose-6-phosphate dehydrogenase immunoprecipitates from radiolabeled uterine tissue extracts previously revealed three proteins: A, B and C, which were tentatively identified as a 60-64 kDa precursor form, a 57 kDa predominant form...

  19. Protective Effect of Quercetin on Oxidative Stress in Glucose-6-Phosphate Dehydrogenase-Deficient Erythrocytes in Vitro

    PubMed Central

    Jamshidzadeh, Akram; Rezaeian Mehrabadi, Abbas

    2010-01-01

    Glucose-6-phosphate dehydrogenase (G6PD) deficient subjects are vulnerable to oxidative stress. Quercetin, a flavonoids, has been employed as a potent oxygen-free radical scavenger in order to assess the protective effects of quercetin against H2O2-induced oxidative damage in G6PD-deficient and normal human erythrocytes. Erythrocytes of G6PD-deficient (n = 10) and normal (n = 10) subjects were incubated with different concentrations of quercetin. The produced thiobarbituric acid reactive substance (TBARS) and glutathione (GSH) level of erythrocytes were then subsequently measured. Different concentrations of quercetin showed no significant hemolysis, compared with the phosphate buffer solution. Upon challenge with H2O2, there was a significant (p < 0.005) decrease in GSH and an increase in TBARS level in G6PD-deficient erythrocytes. With quercetin, it managed to preserve concentrations of 15 to 75 mM preserved GSH and TBARS levels of normal and G6PD-deficient erythrocytes against H2O2-induced oxidative damage. In addition to its well-established antioxidant effects, quercetin was also found to have cytoprotective properties. PMID:24363724

  20. [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

  1. Recombinant sucrose phosphorylase from Leuconostoc mesenteroides: characterization, kinetic studies of transglucosylation, and application of immobilised enzyme for production of alpha-D-glucose 1-phosphate.

    PubMed

    Goedl, Christiane; Schwarz, Alexandra; Minani, Alphonse; Nidetzky, Bernd

    2007-03-30

    Sucrose phosphorylase catalyzes the reversible conversion of sucrose (alpha-D-glucopyranosyl-1,2-beta-D-fructofuranoside) and phosphate into D-fructose and alpha-D-glucose 1-phosphate. We report on the molecular cloning and expression of the structural gene encoding sucrose phosphorylase from Leuconostoc mesenteroides (LmSPase) in Escherichia coli DH10B. The recombinant enzyme, containing an 11 amino acid-long N-terminal metal affinity fusion peptide, was overproduced 60-fold in comparison with the natural enzyme. It was purified to apparent homogeneity using copper-loaded Chelating Sepharose and obtained in 20% yield with a specific activity of 190 Umg(-1). LmSPase was covalently attached onto Eupergit C with a binding efficiency of 50% and used for the continuous production of alpha-D-glucose 1-phosphate from sucrose and phosphate (600 mM each) in a packed-bed immobilised enzyme reactor (30 degrees C, pH 7.0). The reactor was operated at a stable conversion of 91% (550 mM product) and productivity of approximately 11 gl(-1)h(-1) for up to 600 h. A kinetic study of transglucosylation by soluble LmSPase was performed using alpha-d-glucose 1-phosphate as the donor substrate and various alcohols as acceptors. D- and L-arabitol were found to be good glucosyl acceptors. PMID:17215056

  2. 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

  3. 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

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

    PubMed

    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

  5. 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

  6. 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 Central

    2015-01-01

    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

  7. ESR-ENDOR studies of x-irradiated glucose-1-phosphate dipotassium salt

    NASA Astrophysics Data System (ADS)

    Locher, Sarah E.; Box, Harold C.

    1980-01-01

    Single crystals of the dipotassium salt of gluocose-1-phosphate were grown out of water or deuterium oxide and x-irradiated at 4.2 °K. The products of irradiation were identified by means of ESR and ENDOR spectroscopy. In crystals grown out of water, three different alkoxy radicals were observed as oxidation products. In partially deuterated crystals only one of the aforementioned alkoxy radicals was produced. A hydroxyalkyl radical, RHĊOH was also produced by loss of hydrogen from the C6' position. Electrons trapped at intermolecular sites were identified. The distances between the electron and the protons of the polar hydroxy groups forming the trap were deduced from ENDOR measurements and found to be relatively large, namely 1.732 and 1.738 Å.

  8. KlGcr1 controls glucose-6-phosphate dehydrogenase activity and responses to H2O2, cadmium and arsenate in Kluyveromyces lactis.

    PubMed

    Lamas-Maceiras, Mónica; Rodríguez-Belmonte, Esther; Becerra, Manuel; González-Siso, Ma Isabel; Cerdán, Ma Esperanza

    2015-09-01

    It has been previously reported that Gcr1 differentially controls growth and sugar utilization in Saccharomyces cerevisiae and Kluyveromyces lactis, although the regulatory mechanisms causing activation of glycolytic genes are conserved (Neil et al., 2004). We have found that KlGCR1 deletion diminishes glucose consumption and ethanol production, but increases resistance to oxidative stress caused by H2O2, cadmium and arsenate, glucose 6P dehydrogenase activity, and the NADPH/NADP(+) and GSH/GSSG ratios in K. lactis. The gene KlZWF1 that encodes for glucose 6P dehydrogenase, the first enzyme in the pentose phosphate pathway, is transcriptionally regulated by KlGcr1. The high resistance to oxidative stress observed in the ΔKlgcr1 mutant strain, could be explained as a consequence of an increased flux of glucose through the pentose phosphate pathway. Since mitochondrial respiration decreases in the ΔKlgcr1 mutant (García-Leiro et al., 2010), the reoxidation of the NADPH, produced through the pentose phosphate pathway, has to be achieved by the reduction of other molecules implied in the defense against oxidative stress, like GSSG. The higher GSH/GSSG ratio in the mutant would explain its phenotype of increased resistance to oxidative stress. PMID:26164373

  9. Regulatory mechanism of the three-component system HptRSA in glucose-6-phosphate uptake in Staphylococcus aureus.

    PubMed

    Yang, Yifan; Sun, Haipeng; Liu, Xiaoyu; Wang, Mingxing; Xue, Ting; Sun, Baolin

    2016-06-01

    Glucose-6-phosphate (G6P) is a common alternative carbon source for various bacteria, and its uptake usually relies on the hexose phosphate antiporter UhpT. In the human pathogenic bacterium Staphylococcus aureus, the ability to utilize different nutrients, particularly alternative carbon source uptake in glucose-limiting conditions, is essential for its fitness in the host environment during the infectious process. It has been reported that G6P uptake in S. aureus is regulated by the three-component system HptRSA. When G6P is provided as the only carbon source, HptRSA could sense extracellular G6P and activate uhpT expression to facilitate G6P utilization. However, the regulatory mechanism of HptRSA is still unclear. In this study, we further investigated the HptRSA system in S. aureus. First, we confirmed that HptRSA is necessary for the normal growth of this pathogen in chemically defined medium with G6P supplementation, and we discovered that HptRSA could exclusively sense extracellular G6P compared to the other organophosphates we tested. Next, using isothermal titration calorimetry, we found that HptA could bind to G6P, suggesting that it may be the G6P sensor. After that experiment, using an electrophoresis mobility shift assay, we verified that the response regulator HptR could directly bind to the uhpT promoter and identified a putative binding site from -67 to -96-bp. Subsequently, we created different point mutations in the putative binding site and revealed that the entire 30-bp sequence is essential for HptR regulation. In summary, we unveiled the regulatory mechanism of the HptRSA system in S. aureus, HptA most likely functions as the G6P sensor, and HptR could implement its regulatory function by directly binding to a conserved, approximately 30-bp sequence in the uhpT promoter. PMID:26711125

  10. Xylose Isomerase Improves Growth and Ethanol Production Rates from Biomass Sugars for Both Saccharomyces Pastorianus and Saccharomyces Cerevisiae

    PubMed Central

    Miller, Kristen P.; Gowtham, Yogender Kumar; Henson, J. Michael; Harcum, Sarah W.

    2013-01-01

    The demand for biofuel ethanol made from clean, renewable nonfood sources is growing. Cellulosic biomass, such as switch grass (Panicum virgatum L.), is an alternative feedstock for ethanol production; however, cellulosic feedstock hydrolysates contain high levels of xylose, which needs to be converted to ethanol to meet economic feasibility. In this study, the effects of xylose isomerase on cell growth and ethanol production from biomass sugars representative of switch grass were investigated using low cell density cultures. The lager yeast species Saccharomyces pastorianus was grown with immobilized xylose isomerase in the fermentation step to determine the impact of the glucose and xylose concentrations on the ethanol production rates. Ethanol production rates were improved due to xylose isomerase; however, the positive effect was not due solely to the conversion of xylose to xylulose. Xylose isomerase also has glucose isomerase activity, so to better understand the impact of the xylose isomerase on S. pastorianus, growth and ethanol production were examined in cultures provided fructose as the sole carbon. It was observed that growth and ethanol production rates were higher for the fructose cultures with xylose isomerase even in the absence of xylose. To determine whether the positive effects of xylose isomerase extended to other yeast species, a side-by-side comparison of S. pastorianus and Saccharomyces cerevisiae was conducted. These comparisons demonstrated that the xylose isomerase increased ethanol productivity for both the yeast species by increasing the glucose consumption rate. These results suggest that xylose isomerase can contribute to improved ethanol productivity, even without significant xylose conversion. PMID:22866331

  11. Incorporation of /sup 14/C from glucose into amino acids in brain in vitro in the presence of organo- and inorganic lead and pyridoxal phosphate

    SciTech Connect

    Regunathan, S.; Sundaresan, R.

    1983-12-05

    The incorporation of label from U/sup 14/C glucose into glutamic acid, glutamine and GABA remained unaltered with the presence of lead acetate in the medium whereas tetraethyl lead (TEL) affected the incorporation in a characteristic manner in different regions of brain. Glucose uptake however was not influenced by TEL. Pyridoxal phosphate was found to reverse the effect of TEL on the incorporation especially in cerebellum and brainstem but with little effect in cerebral cortex. These findings suggest that the alterations in the GABA metabolism in TEL toxicity could be restored to some extent by pyridoxine in discrete brain areas.

  12. Energy balance-dependent regulation of ovine glucose 6-phosphate dehydrogenase protein isoform expression.

    PubMed

    Triantaphyllopoulos, Kostas A; Laliotis, George P; Bizelis, Iosif A

    2014-01-01

    G6PDH is the rate-limiting enzyme of the pentose phosphate pathway and one of the principal source of NADPH, a major cellular reductant. Importantly, in ruminant's metabolism the aforementioned NADPH provided, is utilized for de novo fatty acid synthesis. Previous work of cloning the ovine (Ovis aries) og6pdh gene has revealed the presence of two cDNA transcripts (og6pda and og6pdb), og6pdb being a product of alternative splicing not similar to any other previously reported.(1) In the current study the effect of energy balance in the ovine G6PDH protein expression was investigated, shedding light on the biochemical features and potential physiological role of the oG6PDB isoform. Changes in energy balance leads to protein expression changes in both transcripts, to the opposite direction and not in a proportional way. Negative energy balance was not in favor of the presence of any particular isoform, while both protein expression levels were not significantly different (P > 0.05). In contrast, at the transition point from negative to positive and on the positive energy balance, there is a significant increase of oG6PDA compared with oG6PDB protein expression (P < 0.001). Both oG6PDH protein isoforms changed significantly toward the positive energy balance. oG6PDA is escalating, while oG6PDB is falling, under the same stimulus (positive energy balance alteration). This change is also positively associated with increasing levels in enzyme activity, 4 weeks post-weaning in ewes' adipose tissue. Furthermore, regression analysis clearly demonstrated the linear correlation of both proteins in response to the WPW, while energy balance, enzyme activity, and oG6PDA relative protein expression follow the same escalating trend; in contrast, oG6PDB relative protein expression falls in time, similar to both transcripts accumulation pattern, as reported previously.(2.) PMID:24575366

  13. 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

  14. Dual Antiplatelet Therapy in Patients with Glucose-6-Phosphate Dehydrogenase Deficiency undergoing PCI with Drug-Eluting Stents.

    PubMed

    Biscaglia, Simone; Ferri, Alessandra; Pavasini, Rita; Campo, Gianluca; Ferrari, Roberto

    2015-01-01

    Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human enzyme defect, affecting more than 400 million people worldwide. In patients with G6PD deficiency, the use of aspirin is controversial, since past studies have reported a potential risk of haemolysis related to its administration, even at low doses. More recent publications have shown that low-dose aspirin administration is safe in these patients. At the same time, no authors have previously reported more than single cases regarding low-dose aspirin treatment in patients with G6PD deficiency undergoing percutaneous coronary intervention (PCI), and most physicians are still sceptical about aspirin administration in these patients. In this paper, we report a case series of five patients with G6PD deficiency receiving PCI with drug-eluting stents (DES) and treatment with dual antiplatelet therapy (DAPT) containing low-dose aspirin, without clinical complications. Moreover, we discuss our internal protocol for managing these patients and provide an overview of the available data. PMID:25843116

  15. Molecular cloning, sequence analysis and expression in Escherichia coli of Camelus dromedarius glucose-6-phosphate dehydrogenase cDNA.

    PubMed

    Saeed, Hesham Mahmoud; Alanazi, Mohammad Saud; Abduljaleel, Zainularifeen; Al-Amri, Abdullah; Khan, Zahid

    2012-06-01

    This study determined the full length sequence of glucose-6-phosphate dehydrogenase cDNA (G6PD) from the Arabian camel Camelus dromedarius using reverse transcription polymerase chain reaction. The C. dromedarius G6PD has an open reading frame of 1545 bp, and the cDNA encodes a protein of 515 amino acid residues with a molecular weight of 59.0 KDa. The amino acid sequence showed the highest identity with Equus caballus (92%) and Homo sapiens (92%). The G6PD cDNA was cloned and expressed into Escherichia coli as a fusion protein and was purified in a single chromatographic step using nickel affinity gel column. The purity and the molecular weight of the enzyme were checked on SDS-PAGE and the purified enzyme showed a single band on the gel with a molecular weight of 63.0 KDa. The specific activity of G6PD was determined to be 289.6 EU/mg protein with a fold purification of 95.45 and yield of 56.8%. PMID:22538316

  16. 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

  17. 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

  18. Hemoglobin E and Glucose-6-Phosphate Dehydrogenase Deficiency and Plasmodium falciparum Malaria in the Chittagong Hill Districts of Bangladesh.

    PubMed

    Shannon, Kerry L; Ahmed, Sabeena; Rahman, Hafizur; Prue, Chai S; Khyang, Jacob; Ram, Malathi; Haq, M Zahirul; Chowdhury, Ashish; Akter, Jasmin; Glass, Gregory E; Shields, Timothy; Nyunt, Myaing M; Khan, Wasif A; Sack, David A; Sullivan, David J

    2015-08-01

    Hemoglobin E is largely confined to south and southeast Asia. The association between hemoglobin E (HbE) and malaria is less clear than that of hemoglobin S and C. As part of a malaria study in the Chittagong Hill Districts of Bangladesh, an initial random sample of 202 individuals showed that 39% and 49% of Marma and Khyang ethnic groups, respectively, were positive for either heterozygous or homozygous hemoglobin E. In this group, 6.4% were also found to be severely deficient and 35% mildly deficient for glucose-6-phosphate dehydrogenase (G6PD). In a separate Plasmodium falciparum malaria case-uninfected control study, the odds of having homozygous hemoglobin E (HbEE) compared with normal hemoglobin (HbAA) were higher among malaria cases detected by passive surveillance than age and location matched uninfected controls (odds ratio [OR] = 5.0, 95% confidence interval [CI] = 1.07-46.93). The odds of heterozygous hemoglobin E (HbAE) compared with HbAA were similar between malaria cases and uninfected controls (OR = 0.71, 95% CI = 0.42-1.19). No association by hemoglobin type was found in the initial parasite density or the proportion parasite negative after 2 days of artemether/lumefantrine treatment. HbEE, but not HbAE status was associated with increased passive case detection of malaria. PMID:26101273

  19. 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

  20. 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

  1. 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. PMID:26017233

  2. 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. PMID:26659432

  3. Red Cell Glucose-6-Phosphate Dehydrogenase Deficiency—A Newly Recognized Cause of Neonatal Jaundice and Kernicterus in Canada

    PubMed Central

    Naiman, J. Lawrence; Kosoy, Martin H.

    1964-01-01

    Seven male newborns of Chinese, Greek and Italian origin presented with severe hemolytic jaundice due to red cell glucose-6-phosphate dehydrogenase (G-6-PD) deficiency. In five, the hemolysis was precipitated by inhalation of mothball vapours in the home. Kernicterus was evident upon admission in six infants and was fatal in four of these. G-6-PD deficiency should be suspected as a cause of jaundice in all full-term male infants of these ethnic groups. The diagnosis can be confirmed in any hospital by the methemoglobin reduction test. In areas similar to Toronto, Canada, where these high-risk ethnic groups prevail, the following measures are recommended: (1) detection of G-6-PD deficient newborns by screening cord bloods of all infants of these ethnic groups; (2) protection of affected infants from potentially hemolytic agents such as naphthalene, certain vitamin K preparations, and sulfonamides; and (3) observation of serum bilirubin levels to assess the need for exchange transfusion for hyperbilirubinemia. ImagesFig. 1 PMID:14226101

  4. African Glucose-6-Phosphate Dehydrogenase Alleles Associated with Protection from Severe Malaria in Heterozygous Females in Tanzania

    PubMed Central

    Manjurano, Alphaxard; Sepulveda, Nuno; Nadjm, Behzad; Mtove, George; Wangai, Hannah; Maxwell, Caroline; Olomi, Raimos; Reyburn, Hugh; Riley, Eleanor M.; Drakeley, Christopher J.; Clark, Taane G.

    2015-01-01

    X-linked Glucose-6-phosphate dehydrogenase (G6PD) A- deficiency is prevalent in sub-Saharan Africa populations, and has been associated with protection from severe malaria. Whether females and/or males are protected by G6PD deficiency is uncertain, due in part to G6PD and malaria phenotypic complexity and misclassification. Almost all large association studies have genotyped a limited number of G6PD SNPs (e.g. G6PD202 / G6PD376), and this approach has been too blunt to capture the complete epidemiological picture. Here we have identified 68 G6PD polymorphisms and analysed 29 of these (i.e. those with a minor allele frequency greater than 1%) in 983 severe malaria cases and controls in Tanzania. We establish, across a number of SNPs including G6PD376, that only female heterozygotes are protected from severe malaria. Haplotype analysis reveals the G6PD locus to be under balancing selection, suggesting a mechanism of protection relying on alleles at modest frequency and avoiding fixation, where protection provided by G6PD deficiency against severe malaria is offset by increased risk of life-threatening complications. Our study also demonstrates that the much-needed large-scale studies of severe malaria and G6PD enzymatic function across African populations require the identification and analysis of the full repertoire of G6PD genetic markers. PMID:25671784

  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. PMID:27597985

  6. Molecular cloning and nucleotide sequence of cDNA for human glucose-6-phosphate dehydrogenase variant A(-)

    SciTech Connect

    Hirono, A.; Beutler, E. )

    1988-06-01

    Glucose-6-phosphate dehydrogenase A(-) is a common variant in Blacks that causes sensitivity to drug- and infection-induced hemolytic anemia. A cDNA library was constructed from Epstein-Barr virus-transformed lymphoblastoid cells from a male who was G6PD A(-). One of four cDNA clones isolated contained a sequence not found in the other clones nor in the published cDNA sequence. Consisting of 138 bases and coding 46 amino acids, this segment of cDNA apparently is derived from the alternative splicing involving the 3{prime} end of intron 7. Comparison of the remaining sequences of these clones with the published sequence revealed three nucleotide substitutions: C{sup 33} {yields} G, G{sup 202} {yields} A, and A{sup 376} {yields} G. Each change produces a new restriction site. Genomic DNA from five G6PD A(-) individuals was amplified by the polymerase chain reaction. The findings of the same mutation in G6PD A(-) as is found in G6PD A(+) strongly suggests that the G6PD A(-) mutation arose in an individual with G6PD A(+), adding another mutation that causes the in vivo instability of this enzyme protein.

  7. 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.

  8. 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

  9. Fatal haemolytic crisis with microvascular pulmonary obstruction mimicking a pulmonary embolism in a young African man with glucose-6-phosphate dehydrogenase deficiency

    PubMed Central

    Albertsen, Jens; Ommen, Hans Beier; Wandler, Anne; Munk, Kim

    2014-01-01

    We report a fatal case of haemolytic crisis mimicking a pulmonary embolism in a previously healthy 42-year-old African man. The patient was admitted to hospital with fatigue, shortness of breath and jaundice lasting for 2 days. Laboratory tests were consistent with haemolysis and inflammation. The patient was treated as having a mycoplasma pneumonia. His condition deteriorated rapidly, with respiratory distress and circulatory failure. Echocardiography showed pulmonary hypertension and right heart dilation. Despite the fact that he was given fibrinolysis for suspected pulmonary embolism, he developed cardiac arrest and died after a long-lasting resuscitation attempt. Postmortem examinations revealed that the patient had a glucose-6-phosphate dehydrogenase deficiency and disseminated intravascular coagulation with pulmonary microthrombi. To the best of our knowledge, this is the first case of death caused by right heart failure due to microvascular obstruction resulting from multiple microvascular thrombosis in a patient with acute haemolysis due to glucose-6-phosphate dehydrogenase deficiency. PMID:24713708

  10. Fermentation of xylulose to ethanol using xylose isomerase and yeasts

    SciTech Connect

    Jeffries, T.W.

    1981-01-01

    In a survey of 35 organisms, predominantly yeasts, about 40% were capable of fermenting xylulose to ethanol. Two species, Candida tropicalis and Schizosaccharomyces pombe, did so at good rates and without an initial lag. Saccharomyces cerevisiae strains that fermented glucose rapidly fermented xylulose at a slower rate. Ten yeasts and three strains of the bacterium Zymomonas mobilis were weak or negative for xylulose, even though they fermented glucose under the conditions employed. C. tropicalis was able to form 1.0 M ethanol from 1.0 M xylose if the fermentation broth was recycled over immobilized xylose isomerase.

  11. 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

  12. Association Studies of Calcium-Sensing Receptor (CaSR) Polymorphisms with Serum Concentrations of Glucose and Phosphate, and Vascular Calcification in Renal Transplant Recipients

    PubMed Central

    Maréchal, Céline; Jadoul, Michel; Devuyst, Olivier; Thakker, Rajesh V.

    2015-01-01

    Background Cardiovascular disease is the major cause of death in renal transplant recipients (RTRs) and linked to arterial calcification. The calcium-sensing receptor (CaSR), a G-protein coupled receptor, plays a pivotal role in extracellular calcium homeostasis and is expressed in the intimal and medial layers of the arterial wall. We investigated whether common CASR gene variants are predictors for aortic and coronary artery calcification or influence risk factors such as serum calcium, phosphate and glucose concentrations in RTRs. Methods Two hundred and eighty four RTRs were investigated for associations between three CASR promoter region single nucleotide polymorphisms (SNPs) (rs115759455, rs7652589, rs1501899), three non-synonymous CASR coding region SNPs (A986S, R990G, Q1011E), and aortic and coronary artery calcium mass scores, cardiovascular outcomes and calcification risk factors that included serum phosphate, calcium, total cholesterol and glucose concentrations. Results Multivariate analysis revealed that RTRs homozygous for the minor allele (SS) of the A986S SNP, when compared to those homozygous for the major allele (AA), had raised serum glucose concentrations (8.7±5.4 vs. 5.7±2.1 mmol/L, P<0.05). In addition, RTRs who were heterozygous (CT) at the rs115759455 SNP, when compared to those homozygous for the major allele (CC), had higher serum phosphate concentrations (1.1±0.3 vs. 1.0±0.2 mmol/L, P<0.05). CASR SNPs were not significant determinants for aortic or coronary artery calcification, and were not associated with cardiovascular outcomes or mortality in this RTR cohort. Conclusions Common CASR SNPs may be independent predictors of serum glucose and phosphate concentrations, but are not determinants of vascular calcification or cardiovascular outcomes. PMID:25786244

  13. INDUCTION AND REPRESSION OF l-ARABINOSE ISOMERASE IN PEDIOCOCCUS PENTOSACEUS1

    PubMed Central

    Dobrogosz, Walter J.; DeMoss, Ralph D.

    1963-01-01

    Dobrogosz, Walter J. (University of Illinois, Urbana) and Ralph D. DeMoss. Induction and repression of l-arabinose isomerase in Pediococcus pentosaceus. J. Bacteriol. 85:1350–1355. 1963.—The inducible l-arabinose isomerase of Pediococcus pentosaceus can be rapidly and conveniently measured in whole-cell preparations by use of a standard colorimetric procedure originally developed for studies with cell-free enzyme preparations. The enzyme is measured by its ability to catalyze the isomerization of l-arabinose to l-ribulose. Whole cells suspended in a suitable buffer and pretreated with toluene were shown to exhibit this isomerase activity at a level comparable with that observed in cell-free enzyme preparations. Conditions for optimal induction of l-arabinose isomerase are described. In addition, it was determined that the formation of this enzyme is subject to repression by glucose, i.e., via catabolite repression. PMID:14047229

  14. Metabolic influence of lead on polyhydroxyalkanoates (PHA) production and phosphate uptake in activated sludge fed with glucose or acetic acid as carbon source.

    PubMed

    You, Sheng-Jie; Tsai, Yung-Pin; Cho, Bo-Chuan; Chou, Yi-Hsiu

    2011-09-01

    Sludge in a sequential batch reactor (SBR) system was used to investigate the effect of lead toxicity on metabolisms of polyphosphate accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs) communities fed with acetic acid or glucose as their sole carbon source, respectively. Results showed that the effect of lead on substrate utilization of both PAOs and GAOs was insignificant. However, lead substantially inhibited both of phosphate release and uptake of PAOs. In high concentration of acetic acid trials, an abnormal aerobic phosphate release was observed instead of phosphate uptake and the release rate increased with increasing lead concentration. Results also showed that PAOs could normally synthesize polyhydroxybutyrate (PHB) in the anaerobic phase even though lead concentration was 40 mg L(-1). However, they could not aerobically utilize PHB normally in the presence of lead. On the other hand, GAOs could not normally metabolize polyhydroxyvalerate (PHV) in both the anaerobic and aerobic phases. PMID:21704513

  15. 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

  16. 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

  17. 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.

  18. Using a Personal Glucose Meter and Alkaline Phosphatase for Point-of-Care Quantification of Galactose-1-Phosphate Uridyltransferase in Clinical Galactosemia Diagnosis.

    PubMed

    Zhang, Jingjing; Xiang, Yu; Novak, Donna E; Hoganson, George E; Zhu, Junjie; Lu, Yi

    2015-10-01

    The personal glucose meter (PGM) was recently shown to be a general meter to detect many targets. Most studies, however, focus on transforming either target binding or enzymatic activity that cleaves an artificial substrate into the production of glucose. More importantly, almost all reports exhibit their methods by using artificial samples, such as buffers or serum samples spiked with the targets. To expand the technology to even broader targets and to validate its potential in authentic, more complex clinical samples, we herein report expansion of the PGM method by using alkaline phosphatase (ALP) that links the enzymatic activity of galactose-1-phosphate uridyltransferase to the production of glucose, which allows point-of-care galactosemia diagnosis in authentic human clinical samples. Given the presence of ALP in numerous enzymatic assays for clinical diagnostics, the methods demonstrated herein advance the field closer to point-of-care detection of a wide range of targets in real clinical samples. PMID:26350570

  19. A comprehensive analysis of membrane and morphology of erythrocytes from patients with glucose-6-phosphate dehydrogenase deficiency.

    PubMed

    Fang, Zishui; Jiang, Chengrui; Tang, Jia; He, Ming; Lin, Xiaoying; Chen, Xiaodan; Han, Luhao; Zhang, Zhiqiang; Feng, Yi; Guo, Yibin; Li, Hongyi; Jiang, Weiying

    2016-06-01

    Acute hemolytic anemia could be triggered by oxidative stress in the patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency. However, the underlying hemolytic mechanism is unknown. To make clear the hemolytic mechanisms, a systematic study on membrane ultrastructure had been undertaken. A comprehensive method was used including atomic force microscopy, scanning electron microscopy, flow cytometer and fluorescence microscopy to analyze the membrane ultrastructure, externalized phosphatidylserine (PS), intracellular Ca(2+) concentration, morphology and the distributions of band 3 protein in G6PD deficient red blood cells (RBCs) after tert-butyl-hydroperoxide (t-BHP) oxidation. The results showed that erythrocyte shrinkage, annexin-V binding to externalized PS on the membrane of early-stage apoptotic cells, the increased membrane roughness and intracellular Ca(2+) concentration, as well as the change of distributions of band 3 protein in RBCs. Compared with the control RBCs, as the concentration of t-BHP up to 0.1mM, the membrane roughness of G6PD deficient RBCs showed significant difference (p<0.05) and as the concentration of t-BHP up to 0.3mM, externalized PS showed significant difference (p<0.05). Furthermore, the population types of RBCs showed dramatic difference between control groups and G6PD deficient groups. Oxidative stress induced more serious erythrocyte apoptosis and resulted in increased roughness of erythrocyte membrane and abnormal distributed band 3 protein in G6PD deficient RBCs. Echinocytes are the predominant abnormal erythrocyte shape occurring in the peripheral blood from patients with G6PD deficiency, which may shorten the RBCs lifespan. The results in the present study will give an increased understanding for the hemolytic mechanism of G6PD deficiency. PMID:26496826

  20. The level of glucose-6-phosphate dehydrogenase activity strongly influences xylose fermentation and inhibitor sensitivity in recombinant Saccharomyces cerevisiae strains.

    PubMed

    Jeppsson, Marie; Johansson, Björn; Jensen, Peter Ruhdal; Hahn-Hägerdal, Bärbel; Gorwa-Grauslund, Marie F

    2003-11-01

    Disruption of the ZWF1 gene encoding glucose-6-phosphate dehydrogenase (G6PDH) has been shown to reduce the xylitol yield and the xylose consumption in the xylose-utilizing recombinant Saccharomyces cerevisiae strain TMB3255. In the present investigation we have studied the influence of different production levels of G6PDH on xylose fermentation. We used a synthetic promoter library and the copper-regulated CUP1 promoter to generate G6PDH-activities between 0% and 179% of the wild-type level. G6PDH-activities of 1% and 6% of the wild-type level resulted in 2.8- and 5.1-fold increase in specific xylose consumption, respectively, compared with the ZWF1-disrupted strain. Both strains exhibited decreased xylitol yields (0.13 and 0.19 g/g xylose) and enhanced ethanol yields (0.36 and 0.34 g/g xylose) compared with the control strain TMB3001 (0.29 g xylitol/g xylose, 0.31 g ethanol/g xylose). Cytoplasmic transhydrogenase (TH) from Azotobacter vinelandii has previously been shown to transfer NADPH and NAD(+) into NADP(+) and NADH, and TH-overproduction resulted in lower xylitol yield and enhanced glycerol yield during xylose utilization. Strains with low G6PDH-activity grew slower in a lignocellulose hydrolysate than the strain with wild-type G6PDH-activity, which suggested that the availability of intracellular NADPH correlated with tolerance towards lignocellulose-derived inhibitors. Low G6PDH-activity strains were also more sensitive to H(2)O(2) than the control strain TMB3001. PMID:14618564

  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. 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. PMID:25925739

  3. Pseudomonas putida KT2440 Strain Metabolizes Glucose through a Cycle Formed by Enzymes of the Entner-Doudoroff, Embden-Meyerhof-Parnas, and Pentose Phosphate Pathways.

    PubMed

    Nikel, Pablo I; Chavarría, Max; Fuhrer, Tobias; Sauer, Uwe; de Lorenzo, Víctor

    2015-10-23

    The soil bacterium Pseudomonas putida KT2440 lacks a functional Embden-Meyerhof-Parnas (EMP) pathway, and glycolysis is known to proceed almost exclusively through the Entner-Doudoroff (ED) route. To investigate the raison d'être of this metabolic arrangement, the distribution of periplasmic and cytoplasmic carbon fluxes was studied in glucose cultures of this bacterium by using (13)C-labeled substrates, combined with quantitative physiology experiments, metabolite quantification, and in vitro enzymatic assays under both saturating and non-saturating, quasi in vivo conditions. Metabolic flux analysis demonstrated that 90% of the consumed sugar was converted into gluconate, entering central carbon metabolism as 6-phosphogluconate and further channeled into the ED pathway. Remarkably, about 10% of the triose phosphates were found to be recycled back to form hexose phosphates. This set of reactions merges activities belonging to the ED, the EMP (operating in a gluconeogenic fashion), and the pentose phosphate pathways to form an unforeseen metabolic architecture (EDEMP cycle). Determination of the NADPH balance revealed that the default metabolic state of P. putida KT2440 is characterized by a slight catabolic overproduction of reducing power. Cells growing on glucose thus run a biochemical cycle that favors NADPH formation. Because NADPH is required not only for anabolic functions but also for counteracting different types of environmental stress, such a cyclic operation may contribute to the physiological heftiness of this bacterium in its natural habitats. PMID:26350459

  4. Delineation of the roles of amino acids involved in the catalytic functions of Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase.

    PubMed

    Vought, V; Ciccone, T; Davino, M H; Fairbairn, L; Lin, Y; Cosgrove, M S; Adams, M J; Levy, H R

    2000-12-12

    The roles of particular amino acids in substrate and coenzyme binding and catalysis of glucose-6-phosphate dehydrogenase of Leuconostoc mesenteroides have been investigated by site-directed mutagenesis, kinetic analysis, and determination of binding constants. The enzyme from this species has functional dual NADP(+)/NAD(+) specificity. Previous investigations in our laboratories determined the three-dimensional structure. Kinetic studies showed an ordered mechanism for the NADP-linked reaction while the NAD-linked reaction is random. His-240 was identified as the catalytic base, and Arg-46 was identified as important for NADP(+) but not NAD(+) binding. Mutations have been selected on the basis of the three-dimensional structure. Kinetic studies of 14 mutant enzymes are reported and kinetic mechanisms are reported for 5 mutant enzymes. Fourteen substrate or coenzyme dissociation constants have been measured for 11 mutant enzymes. Roles of particular residues are inferred from k(cat), K(m), k(cat)/K(m), K(d), and changes in kinetic mechanism. Results for enzymes K182R, K182Q, K343R, and K343Q establish Lys-182 and Lys-343 as important in binding substrate both to free enzyme and during catalysis. Studies of mutant enzymes Y415F and Y179F showed no significant contribution for Tyr-415 to substrate binding and only a small contribution for Tyr-179. Changes in kinetics for T14A, Q47E, and R46A enzymes implicate these residues, to differing extents, in coenzyme binding and discrimination between NADP(+) and NAD(+). By the same measure, Lys-343 is also involved in defining coenzyme specificity. Decrease in k(cat) and k(cat)/K(m) for the D374Q mutant enzyme defines the way Asp-374, unique to L. mesenteroides G6PD, modulates stabilization of the enzyme during catalysis by its interaction with Lys-182. The greatly reduced k(cat) values of enzymes P149V and P149G indicate the importance of the cis conformation of Pro-149 in accessing the correct transition state. PMID

  5. 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

  6. Prevalence and molecular basis of glucose-6-phosphate dehydrogenase deficiency in Afghan populations: implications for treatment policy in the region

    PubMed Central

    2013-01-01

    Background Glucose-6-phosphate dehydrogenase deficiency (G6PD), an x-linked inherited enzymopathy, is a barrier to malaria control because primaquine cannot be readily applied for radical cure in individuals with the condition. In endemic areas, including in Afghanistan, the G6PD status of vivax patients is not routinely determined so the drug is rarely, if ever, prescribed even though it is included as a recommended treatment in local, regional and global guidelines. This study assessed the prevalence and genotype of G6PD deficiency in Afghan populations and examined the need for routine G6PD testing as a malaria treatment and control tool. Methods A cross-sectional household survey was conducted using random sampling in five Afghan cities to determine the prevalence of G6PD deficiency in Afghan ethnic groups. Filter-paper blood spots were analysed for phenotypic G6PD deficiency using a fluorescent spot test. Molecular analysis was conducted to identify the genetic basis of the disorder. Results Overall, 45/1,436 (3.1%) people were G6PD deficient, 36/728 (5.0%) amongst males and 9/708 (1.3%) amongst females. Amongst males the prevalence was highest in the Pashtun ethnic group (10%, 26/260) while in Tajik males it was 8/250 (3.2%); in Hazara males it was 1/77 (1.3%) and in Uzbek males is was 0/125. Genetic testing in those with deficiency showed that all were of the Mediterranean type (Med-) characterized by a C-T change at codon 563 of the G6PD gene. Conclusion Prevalence of G6PD deficiency in Afghanistan varies considerably by ethnic group and is predominantly of the Mediterranean type. G6PD deficient individuals are susceptible to potentially severe and life-threatening haemolysis after standard primaquine treatment. If the aim of increasing access to radical treatment of vivax is to be successful reliable G6PD testing needs to be made routinely available within the health system. PMID:23834949

  7. The crystal and solution studies of glucosamine-6-phosphate synthase from Candida albicans.

    PubMed

    Raczynska, Joanna; Olchowy, Jaroslaw; Konariev, Peter V; Svergun, Dmitri I; Milewski, Slawomir; Rypniewski, Wojciech

    2007-09-21

    Glucosamine 6-phosphate (GlcN-6-P) synthase is an ubiquitous enzyme that catalyses the first committed step in the reaction pathway that leads to formation of uridine 5'-diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc), a precursor of macromolecules that contain amino sugars. Despite sequence similarities, the enzyme in eukaryotes is tetrameric, whereas in prokaryotes it is a dimer. The activity of eukaryotic GlcN-6-P synthase (known as Gfa1p) is regulated by feedback inhibition by UDP-GlcNAc, the end product of the reaction pathway, whereas in prokaryotes the GlcN-6-P synthase (known as GlmS) is not regulated at the post-translational level. In bacteria and fungi the enzyme is essential for cell wall synthesis. In human the enzyme is a mediator of insulin resistance. For these reasons, Gfa1p is a target in anti-fungal chemotherapy and in therapeutics for type-2 diabetes. The crystal structure of the Gfa1p isomerase domain from Candida albicans has been analysed in complex with the allosteric inhibitor UDP-GlcNAc and in the presence of glucose 6-phosphate, fructose 6-phosphate and an analogue of the reaction intermediate, 2-amino-2-deoxy-d-mannitol 6-phosphate (ADMP). A solution structure of the native Gfa1p has been deduced using small-angle X-ray scattering (SAXS). The tetrameric Gfa1p can be described as a dimer of dimers, with each half similar to the related enzyme from Escherichia coli. The core of the protein consists of the isomerase domains. UDP-GlcNAc binds, together with a metal cation, in a well-defined pocket on the surface of the isomerase domain. The residues responsible for tetramerisation and for binding UDP-GlcNAc are conserved only among eukaryotic sequences. Comparison with the previously studied GlmS from E. coli reveals differences as well as similarities in the isomerase active site. This study of Gfa1p focuses on the features that distinguish it from the prokaryotic homologue in terms of quaternary structure, control of the enzymatic activity

  8. CpsE from Type 2 Streptococcus pneumoniae Catalyzes the Reversible Addition of Glucose-1-Phosphate to a Polyprenyl Phosphate Acceptor, Initiating Type 2 Capsule Repeat Unit Formation

    PubMed Central

    Cartee, Robert T.; Forsee, W. Thomas; Bender, Matthew H.; Ambrose, Karita D.; Yother, Janet

    2005-01-01

    The majority of the 90 capsule types made by the gram-positive pathogen Streptococcus pneumoniae are assembled by a block-type mechanism similar to that utilized by the Wzy-dependent O antigens and capsules of gram-negative bacteria. In this mechanism, initiation of repeat unit formation occurs by the transfer of a sugar to a lipid acceptor. In S. pneumoniae, this step is catalyzed by CpsE, a protein conserved among the majority of capsule types. Membranes from S. pneumoniae type 2 strain D39 and Escherichia coli containing recombinant Cps2E catalyzed incorporation of [14C]Glc from UDP-[14C]Glc into a lipid fraction in a Cps2E-dependent manner. The Cps2E-dependent glycolipid product from both membranes was sensitive to mild acid hydrolysis, suggesting that Cps2E was catalyzing the formation of a polyprenyl pyrophosphate Glc. Addition of exogenous polyprenyl phosphates ranging in size from 35 to 105 carbons to D39 and E. coli membranes stimulated Cps2E activity. The stimulation was due, in part, to utilization of the exogenous polyprenyl phosphates as an acceptor. The glycolipid product synthesized in the absence of exogenous polyprenyl phosphates comigrated with a 60-carbon polyprenyl pyrophosphate Glc. When 10 or 100 μM UMP was added to reaction mixtures containing D39 membranes, Cps2E activity was inhibited 40% and 80%, respectively. UMP, which acted as a competitive inhibitor of UDP-Glc, also stimulated Cps2E to catalyze the reverse reaction, with synthesis of UDP-Glc from the polyprenyl pyrophosphate Glc. These data indicated that Cps2E was catalyzing the addition of Glc-1-P to a polyprenyl phosphate acceptor, likely undecaprenyl phosphate. PMID:16237026

  9. In vivo and in vitro effects of some plant hormones on rat erythrocyte carbonic anhydrase and glucose-6-phosphate dehydrogenase activities.

    PubMed

    Ciftçi, Mehmet; Demir, Yavuz; Ozmen, Ismail; Atici, Okkeş

    2003-02-01

    The present study was undertaken to determine in vivo and in vitro effects of some plant growth regulators on rat erythrocyte carbonic anhydrase (CA) and glucose-6-phosphate dehydrogenase (G6PD) activities. Both in vivo and in vitro, spermidine and kinetin did not affect enzymatic activities of CA and G6PD, whereas putrescine decreased these activities, and abscisic acid increased them. Since plants use such growth regulators, their effects should be considered on mammals consuming them since they may possess important biological effects. PMID:12751824

  10. 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

  11. Calculation of the pentose phosphate and Embden-Myerhoff pathways from a single incubation with (U-/sup 14/C)- and (5-/sup 3/H)glucose

    SciTech Connect

    O'Fallon, J.V.; Wright, R.W. Jr.

    1987-04-01

    A method that simultaneously determines Embden-Myerhoff pathway and pentose phosphate pathway (PPP) activities from an incubation with (U-/sup 14/C)- and (5-/sup 3/H)glucose is presented. The method relies on the use of unlabeled pyruvate and lactate to dilute out radiolabel entering the tricarboxylic acid cycle. Gluconeogenesis from pyruvate is prevented by the use of an incubation chamber that maintains a CO/sub 2/ (and bicarbonate) free environment. The method, which includes the contribution by the recycling steps of the PPP, is especially useful when biological material is limited or developmental timing is critical.

  12. Ribose metabolism and nucleic acid synthesis in normal and glucose-6-phosphate dehydrogenase-deficient human erythrocytes infected with Plasmodium falciparum.

    PubMed Central

    Roth, E F; Ruprecht, R M; Schulman, S; Vanderberg, J; Olson, J A

    1986-01-01

    The metabolism of pentose-phosphate was investigated in Plasmodium falciparum-infected normal and glucose-6-phosphate dehydrogenase (G6PD)-deficient human red blood cells in vitro. 5'-Phosphoribosyl-1-pyrophosphate (PRPP) content of infected normal red blood cells was increased 50-60-fold at the parasite trophozoite growth stage over that of uninfected cells. The PRPP increment in infected G6PD-deficient cells at comparable stage and parasitemia was only 40% of the value in normal infected cells. Red blood cell PRPP synthetase activity did not change during the growth cycle of the parasite and was similar in both normal and G6PD-deficient cells. Reduced glutathione (GSH) content of G6PD-deficient cells under conditions of culture fell to low or undetectable levels. These low levels of GSH were shown to inhibit the function of red blood cell PRPP synthetase, which requires GSH for full activity. Measurements of the incorporation of 1-14C or 6-14C selectively labeled glucose into parasite nucleic acids revealed that in normal infected red cells, approximately 20% of the pentose was produced via the oxidation of glucose-6-phosphate, whereas in infected G6PD-deficient cells (Mediterranean type), none of the pentose was produced via the oxidative pathway. It is concluded that the low level of reduced GSH found in G6PD deficiency and the resultant partial inhibition of PRPP synthetase together with the missing oxidative pathway for ribose phosphate production can account fully for the reduced parasite growth rate in G6PD-deficient red blood cells described previously. Of these two mechanisms, the predominant one is the impaired PRPP synthetase activity due to low GSH levels in enzyme-deficient red blood cells. The contribution to the ribose-phosphate pool by the hexose monophosphate shunt is relatively minor. A co-existing oxidative stress (which is often hypothesized to mediate the destruction of parasitized red blood cells) is not required to explain growth inhibition

  13. Co-production of hydrogen and ethanol from glucose by modification of glycolytic pathways in Escherichia coli - from Embden-Meyerhof-Parnas pathway to pentose phosphate pathway.

    PubMed

    Seol, Eunhee; Sekar, Balaji Sundara; Raj, Subramanian Mohan; Park, Sunghoon

    2016-02-01

    Hydrogen (H2) production from glucose by dark fermentation suffers from the low yield. As a solution to this problem, co-production of H2 and ethanol, both of which are good biofuels, has been suggested. To this end, using Escherichia coli, activation of pentose phosphate (PP) pathway, which can generate more NADPH than the Embden-Meyhof-Parnas (EMP) pathway, was attempted. Overexpression of two key enzymes in the branch nodes of the glycolytic pathway, Zwf and Gnd, significantly improved the co-production of H2 and ethanol with concomitant reduction of pyruvate secretion. Gene expression analysis and metabolic flux analysis (MFA) showed that, upon overexpression of Zwf and Gnd, glucose assimilation through the PP pathway, compared with that of the EMP or Entner-Doudoroff (ED) pathway, was greatly enhanced. The maximum co-production yields were 1.32 mol H2 mol(-1) glucose and 1.38 mol ethanol mol(-1) glucose, respectively. It is noteworthy that the glycolysis and the amount of NAD(P)H formed under anaerobic conditions could be altered by modifying (the activity of) several key enzymes. Our strategy could be applied for the development of industrial strains for biological production of reduced chemicals and biofuels which suffers from lack of reduced co-factors. PMID:26581029

  14. Comparing the xylose reductase/xylitol dehydrogenase and xylose isomerase pathways in arabinose and xylose fermenting Saccharomyces cerevisiae strains

    PubMed Central

    Bettiga, Maurizio; Hahn-Hägerdal, Bärbel; Gorwa-Grauslund, Marie F

    2008-01-01

    Background Ethanolic fermentation of lignocellulosic biomass is a sustainable option for the production of bioethanol. This process would greatly benefit from recombinant Saccharomyces cerevisiae strains also able to ferment, besides the hexose sugar fraction, the pentose sugars, arabinose and xylose. Different pathways can be introduced in S. cerevisiae to provide arabinose and xylose utilisation. In this study, the bacterial arabinose isomerase pathway was combined with two different xylose utilisation pathways: the xylose reductase/xylitol dehydrogenase and xylose isomerase pathways, respectively, in genetically identical strains. The strains were compared with respect to aerobic growth in arabinose and xylose batch culture and in anaerobic batch fermentation of a mixture of glucose, arabinose and xylose. Results The specific aerobic arabinose growth rate was identical, 0.03 h-1, for the xylose reductase/xylitol dehydrogenase and xylose isomerase strain. The xylose reductase/xylitol dehydrogenase strain displayed higher aerobic growth rate on xylose, 0.14 h-1, and higher specific xylose consumption rate in anaerobic batch fermentation, 0.09 g (g cells)-1 h-1 than the xylose isomerase strain, which only reached 0.03 h-1 and 0.02 g (g cells)-1h-1, respectively. Whereas the xylose reductase/xylitol dehydrogenase strain produced higher ethanol yield on total sugars, 0.23 g g-1 compared with 0.18 g g-1 for the xylose isomerase strain, the xylose isomerase strain achieved higher ethanol yield on consumed sugars, 0.41 g g-1 compared with 0.32 g g-1 for the xylose reductase/xylitol dehydrogenase strain. Anaerobic fermentation of a mixture of glucose, arabinose and xylose resulted in higher final ethanol concentration, 14.7 g l-1 for the xylose reductase/xylitol dehydrogenase strain compared with 11.8 g l-1 for the xylose isomerase strain, and in higher specific ethanol productivity, 0.024 g (g cells)-1 h-1 compared with 0.01 g (g cells)-1 h-1 for the xylose reductase

  15. Genetics Home Reference: triosephosphate isomerase deficiency

    MedlinePlus

    ... of triosephosphate isomerase deficiency. Eur J Haematol. 2011 Mar;86(3):265-7. doi: 10.1111/j.1600-0609.2010.01484.x. Citation on PubMed Orosz F, Oláh J, Ovádi J. Triosephosphate isomerase deficiency: facts and doubts. IUBMB Life. 2006 Dec;58(12):703-15. Review. Citation ...

  16. Urinary loss of glucose, phosphate, and protein by diffusion into proximal straight tubules injured by D-serine and maleic acid

    SciTech Connect

    Carone, F.A.; Nakamura, S.; Goldman, B.

    1985-06-01

    In several models of acute renal failure leakage of glomerular filtrate out of the tubule is an important pathogenetic mechanism; however, bidirectional diffusion of solute to account for certain pathophysiologic features of acute renal failure has received meager attention. Using micropuncture and clearance methods, the authors assessed sequentially leakage of solutes and inulin across proximal straight tubules (PST) injured by two nephrotoxins. In d-serine-treated rats with extensive necrosis of PST, the basis for glucosuria and tubular leakage of inulin was studied. Glucose absorption by the proximal convoluted tubule and glucose delivery to the PST were normal, but glucose delivery to the distal tubule was increased nearly 8-fold, indicating diffusion of glucose from interstitial to tubular luminal fluid across the necrotic PST. Total kidney inulin clearance was greatly reduced, but single nephron glomerular filtration rate, based on proximal convoluted tubule samples, was normal, indicating tubular loss of inulin. Urinary recovery of (/sup 14/C)inulin infused into tubular lumina revealed that proximal convoluted tubule and distal tubule were impermeable to inulin and that inulin diffused out of the necrotic PST. The progressive return over 6 days of tubular impermeability for inulin correlated with relining of PST with new cells. In maleic acid-treated rats the site and extent of tubular necrosis and the nature of urinary loss of solutes were studied. Microdissection revealed that maleic acid caused limited necrosis of PST which averaged 7.4% of total proximal tubular length. Increased urinary excretion of protein, phosphate, and glucose and increased tubular permeability to microinfused (/sup 14/C)inulin occurred with the onset of PST necrosis, and return of these abnormalities to normal correlated with the degree of cellular repair of the PST.

  17. Identification of an arginine residue in the dual coenzyme-specific glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides that plays a key role in binding NADP+ but not NAD+.

    PubMed

    Levy, H R; Vought, V E; Yin, X; Adams, M J

    1996-02-01

    Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides can utilize either NADP or NAD as coenzyme. The enzyme's three-dimensional structure has been solved (Rowland et al., 1994, Structure 2, 1073-1087) and shown to contain a conventional nucleotide binding domain. NADP+ was modeled into the structure by superimposing the beta alpha beta domain and that of coenzyme-bound 6-phosphogluconate dehydrogenase (Adams et al., 1994, Structure 2, 651-658), enabling us to identify Arg-46 as a potentially important residue for NADP+ binding. Using site-directed mutagenesis, we constructed mutant enzymes in which Arg-46 was replaced by glutamine (R46Q) and alanine (R46A) and examined their kinetic properties. The principal effects in these mutant enzymes were that the Km and Ki values for NADP+ increased by 2 to 3 orders of magnitude over those of the wild-type enzyme. No other kinetic constant was altered more than 6.5-fold. Changing this single amino acid leads to mutant glucose-6-phosphate dehydrogenases with coenzyme specificities that favor NAD+, whereas the wild-type enzyme prefers NADP+ as coenzyme. These results confirm that Arg-46 plays a key role in NADP+ binding by contributing a positively charged planar residue that interacts primarily with the 2'-adenosine phosphate. The Arg residue corresponding to Arg-46 in L. mesenteroides glucose-6-phosphate dehydrogenase is conserved in all glucose-6-phosphate dehydrogenases and, presumably, plays the same role in all these enzymes. PMID:8579362

  18. Yihx-encoded haloacid dehalogenase-like phosphatase HAD4 from Escherichia coli is a specific α-d-glucose 1-phosphate hydrolase useful for substrate-selective sugar phosphate transformations

    PubMed Central

    Pfeiffer, Martin; Wildberger, Patricia; Nidetzky, Bernd

    2014-01-01

    Phosphomonoester hydrolases (phosphatases; EC 3.1.3.) often exhibit extremely relaxed substrate specificity which limits their application to substrate-selective biotransformations. In search of a phosphatase catalyst specific for hydrolyzing α-d-glucose 1-phosphate (αGlc 1-P), we selected haloacid dehalogenase-like phosphatase 4 (HAD4) from Escherichia coli and obtained highly active recombinant enzyme through a fusion protein (Zbasic2_HAD4) that contained Zbasic2, a strongly positively charged three α-helical bundle module, at its N-terminus. Highly pure Zbasic2_HAD4 was prepared directly from E. coli cell extract using capture and polishing combined in a single step of cation exchange chromatography. Kinetic studies showed Zbasic2_HAD4 to exhibit 565-fold preference for hydrolyzing αGlc 1-P (kcat/KM = 1.87 ± 0.03 mM−1 s−1; 37 °C, pH 7.0) as compared to d-glucose 6-phosphate (Glc 6-P). Also among other sugar phosphates, αGlc 1-P was clearly preferred. Using different mixtures of αGlc 1-P and Glc 6-P (e.g. 180 mM each) as the substrate, Zbasic2_HAD4 could be used to selectively convert the αGlc 1-P present, leaving back all of the Glc 6-P for recovery. Zbasic2_HAD4 was immobilized conveniently using direct loading of E. coli cell extract on sulfonic acid group-containing porous carriers, yielding a recyclable heterogeneous biocatalyst that was nearly as effective as the soluble enzyme, probably because protein attachment to the anionic surface occurred in a preferred orientation via the cationic Zbasic2 module. Selective removal of αGlc 1-P from sugar phosphate preparations could be an interesting application of Zbasic2_HAD4 for which readily available broad-spectrum phosphatases are unsuitable. PMID:25484615

  19. A Tale of Two Isomerases: Compact versus Extended Active Sites in Ketosteroid Isomerase and Phosphoglucose Isomerase

    SciTech Connect

    Somarowthu, Srinivas; Brodkin, Heather R.; D’Aquino, J. Alejandro; Ringe, Dagmar; Ondrechen, Mary Jo; Beuning, Penny J.

    2012-07-11

    Understanding the catalytic efficiency and specificity of enzymes is a fundamental question of major practical and conceptual importance in biochemistry. Although progress in biochemical and structural studies has enriched our knowledge of enzymes, the role in enzyme catalysis of residues that are not nearest neighbors of the reacting substrate molecule is largely unexplored experimentally. Here computational active site predictors, THEMATICS and POOL, were employed to identify functionally important residues that are not in direct contact with the reacting substrate molecule. These predictions then guided experiments to explore the active sites of two isomerases, Pseudomonas putida ketosteroid isomerase (KSI) and human phosphoglucose isomerase (PGI), as prototypes for very different types of predicted active sites. Both KSI and PGI are members of EC 5.3 and catalyze similar reactions, but they represent significantly different degrees of remote residue participation, as predicted by THEMATICS and POOL. For KSI, a compact active site of mostly first-shell residues is predicted, but for PGI, an extended active site in which residues in the first, second, and third layers around the reacting substrate are predicted. Predicted residues that have not been previously tested experimentally were investigated by site-directed mutagenesis and kinetic analysis. In human PGI, single-point mutations of the predicted second- and third-shell residues K362, H100, E495, D511, H396, and Q388 show significant decreases in catalytic activity relative to that of the wild type. The results of these experiments demonstrate that, as predicted, remote residues are very important in PGI catalysis but make only small contributions to catalysis in KSI.

  20. Triosephosphate isomerase: energetics of the reaction catalyzed by the yeast enzyme expressed in Escherichia coli

    SciTech Connect

    Nickbarg, E.B.; Knowles, J.R.

    1988-08-09

    Triosephosphate isomerase from bakers' yeast, expressed in Escherichia coli strain DF502(p12), has been purified to homogeneity. The kinetics of the reaction in each direction have been determined at pH 7.5 and 30 degrees C. Deuterium substitution at the C-2 position of substrate (R)-glyceraldehyde phosphate and at the 1-pro-R position of substrate dihydroxyacetone phosphate results in kinetic isotope effects on kcat of 1.6 and 3.4, respectively. The extent of transfer of tritium from (1(R)-TH)dihydroxyacetone phosphate to product (R)-glyceraldehyde phosphate during the catalyzed reaction is only 3% after 66% conversion to product, indicating that the enzymic base that mediates proton transfer is in rapid exchange with solvent protons. When the isomerase-catalyzed reaction is run in tritiated water in each direction, radioactivity is incorporated both into the remaining substrate and into the product. In the exchange-conversion experiment with dihydroxyacetone phosphate as substrate, the specific radioactivity of remaining dihydroxyacetone phosphate rises as a function of the extent of reaction with a slope of about 0.3, while the specific radioactivity of the products is 54% that of the solvent. In the reverse direction with (R)-glyceraldehyde phosphate as substrate, the specific radioactivity of the product formed is only 11% that of the solvent, while the radioactivity incorporated into the remaining substrate (R)-glyceraldehyde phosphate also rises as a function of the extent of reaction with a slope of 0.3. These results have been analyzed according to the protocol described earlier to yield the free energy profile of the reaction catalyzed by the yeast isomerase.

  1. Loss of Cytosolic Phosphoglucose Isomerase Affects Carbohydrate Metabolism in Leaves and Is Essential for Fertility of Arabidopsis1[C][W][OPEN

    PubMed Central

    Kunz, Hans-Henning; Zamani-Nour, Shirin; Häusler, Rainer E.; Ludewig, Katja; Schroeder, Julian I.; Malinova, Irina; Fettke, Joerg; Flügge, Ulf-Ingo; Gierth, Markus

    2014-01-01

    Carbohydrate metabolism in plants is tightly linked to photosynthesis and is essential for energy and carbon skeleton supply of the entire organism. Thus, the hexose phosphate pools of the cytosol and the chloroplast represent important metabolic resources that are maintained through action of phosphoglucose isomerase (PGI) and phosphoglucose mutase interconverting glucose 6-phosphate, fructose 6-phosphate, and glucose 1-phosphate. Here, we investigated the impact of disrupted cytosolic PGI (cPGI) function on plant viability and metabolism. Overexpressing an artificial microRNA targeted against cPGI (amiR-cpgi) resulted in adult plants with vegetative tissue essentially free of cPGI activity. These plants displayed diminished growth compared with the wild type and accumulated excess starch in chloroplasts but maintained low sucrose content in leaves at the end of the night. Moreover, amiR-cpgi plants exhibited increased nonphotochemical chlorophyll a quenching during photosynthesis. In contrast to amiR-cpgi plants, viable transfer DNA insertion mutants disrupted in cPGI function could only be identified as heterozygous individuals. However, homozygous transfer DNA insertion mutants could be isolated among plants ectopically expressing cPGI. Intriguingly, these plants were only fertile when expression was driven by the ubiquitin10 promoter but sterile when the seed-specific unknown seed protein promoter or the Cauliflower mosaic virus 35S promoter were employed. These data show that metabolism is apparently able to compensate for missing cPGI activity in adult amiR-cpgi plants and indicate an essential function for cPGI in plant reproduction. Moreover, our data suggest a feedback regulation in amiR-cpgi plants that fine-tunes cytosolic sucrose metabolism with plastidic starch turnover. PMID:25104722

  2. 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.

  3. Site directed immobilization of glucose-6-phosphate dehydrogenase via thiol-disulfide interchange: influence on catalytic activity of cysteines introduced at different positions.

    PubMed

    Simons, J R; Mosisch, M; Torda, A E; Hilterhaus, L

    2013-08-10

    This study shows the effect of site-directed enzyme immobilization upon the enzyme activity of covalently bound glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides. Immobilization points were introduced at sterically accessible sites in order to control the protein's orientation and twice as much activity was recovered in comparison to conventionally immobilized enzyme. Immobilization of G6PDH via genetically engineered cysteine provided a simple, but effective method to control the immobilization process. G6PDH variants with cysteine close to the active center (L218C), close to the dimer interface (D205C) as well as far from the active center (D453C) showed changes in activity and the efficacy of immobilization. PMID:23770076

  4. A Novel de novo Mutation in the G6PD Gene in a Korean Boy with Glucose-6-phosphate Dehydrogenase Deficiency: Case Report.

    PubMed

    Jang, Mi-Ae; Kim, Ji-Yoon; Lee, Ki-O; Kim, Sun-Hee; Koo, Hong Hoe; Kim, Hee-Jin

    2015-01-01

    Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked recessive hemolytic anemia caused by a mutation in the G6PD gene on Xq28. Herein, we describe a Korean boy with G6PD deficiency resulting from a novel mutation in G6PD. A 20-month-old boy with hemolytic anemia was referred for molecular diagnosis. He had no relevant family history. The G6PD activity was severely decreased at 0.2 U/g Hb (severe deficiency). Direct sequencing analyses on the G6PD gene revealed that he was hemizygous for a novel missense variant, c.1187C>G (p.Pro396Arg), in exon 10 of G6PD. Family study involving his parents revealed the de novo occurrence of the mutation. This is the first report of genetically confirmed G6PD deficiency in Korea. PMID:26275698

  5. Crystallization and preliminary X-ray characterization of phosphoglucose isomerase from Mycobacterium tuberculosis H37Rv

    PubMed Central

    Mathur, Divya; Anand, Kanchan; Mathur, Deepika; Jagadish, Nirmala; Suri, Anil; Garg, Lalit C.

    2007-01-01

    Phosphoglucose isomerase is a ubiquitous enzyme that catalyzes the isomerization of d-glucopyranose-6-phosphate to d-fructofuranose-6-phosphate. The present investigation reports the expression, purification, crystallization and preliminary crystallographic studies of the phosphoglucose isomerase from Mycobacterium tuberculosis H37Rv, which shares 46% sequence identity with that of its human host. The recombinant protein, which was prepared using an Escherichia coli expression system, was crystallized by the hanging-drop vapour-diffusion method. The crystals diffracted to a resolution of 2.8 Å and belonged to the orthorhombic space group I212121, with unit-cell parameters a = 109.0, b = 119.8, c = 138.9 Å. PMID:17401215

  6. Increased basal oxidation of peroxiredoxin 2 and limited peroxiredoxin recycling in glucose-6-phosphate dehydrogenase-deficient erythrocytes from newborn infants.

    PubMed

    Cheah, Fook-Choe; Peskin, Alexander V; Wong, Fei-Liang; Ithnin, Azlin; Othman, Ainoon; Winterbourn, Christine C

    2014-07-01

    Erythrocytes require glucose-6-phosphate dehydrogenase (G6PD) to generate NADPH and protect themselves against hemolytic anemia induced by oxidative stress. Peroxiredoxin 2 (Prx2) is a major antioxidant enzyme that requires NADPH to recycle its oxidized (disulfide-bonded) form. Our aims were to determine whether Prx2 is more highly oxidized in G6PD-deficient erythrocytes and whether these cells are able to recycle oxidized Prx2 after oxidant challenge. Blood was obtained from 61 Malaysian neonates with G6PD deficiency (average 33% normal activity) and 86 controls. Prx2 redox state was analyzed by Western blotting under nonreducing conditions. Prx2 in freshly isolated blood was predominantly reduced in both groups, but the median level of oxidation was significantly higher (8 vs 3%) and the range greater for the G6PD-deficient population. When treated with reagent H2O2, the G6PD-deficient erythrocytes were severely compromised in their ability to recycle oxidized Prx2, with only 27 or 4% reduction after 1 h treatment with 0.1 or 1 mM H2O2 respectively, compared with >97% reduction in control erythrocytes. The accumulation of oxidized Prx2 in oxidatively stressed erythrocytes with common G6PD variants suggests that impaired antioxidant activity of Prx2 could contribute to the hemolysis and other complications associated with the condition.-Cheah, F.-C., Peskin, A. V., Wong, F.-L., Ithnin, A., Othman, A., Winterbourn, C. C. Increased basal oxidation of peroxiredoxin 2 and limited peroxiredoxin recycling in glucose-6-phosphate dehydrogenase deficient erythrocytes from newborn infants. PMID:24636884

  7. 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

  8. Automated Yeast Transformation Protocol to Engineer S. cerevisiae Strains for Cellulosic Ethanol Production with Open Reading Frames that Express Proteins Binding to Xylose Isomerase Identified using Robotic Two-hybrid Screen

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Commercialization of fuel ethanol production from lignocellulosic biomass has focused on engineering the glucose-fermenting industrial yeast Saccharomyces cerevisiae to utilize pentose sugars. Since S. cerevisiae naturally metabolizes xylulose, one approach involves introducing xylose isomerase (XI...

  9. Triosephosphate isomerase: removal of a putatively electrophilic histidine residue results in a subtle change in catalytic mechanism

    SciTech Connect

    Nickbarg, E.B.; Davenport, R.C.; Petsko, G.A.; Knowles, J.R.

    1988-08-09

    An important active-site residue in the glycolytic enzyme triosephosphate isomerase is His-95, which appears to act as an electrophilic component in catalyzing the enolization of the substrates. With the techniques of site-directed mutagenesis, His-95 has been replaced by Gln in the isomerase from Saccharomyces cerevisiae. The mutant isomerase has been expressed in Escherichia coli strain DF502 and purified to homogeneity. The specific catalytic activity of the mutant enzyme is less than that of wild type by a factor of nearly 400. The mutant enzyme can be resolved from the wild-type isomerase on nondenaturing isoelectric focusing gels, and an isomerase activity stain shows that the observed catalytic activity indeed derives from the mutant protein. The mutant enzyme shows the same stereospecificity of proton transfer as the wild type. Tritium exchange experiments similar to those used to define the free energy profile for the wild-type yeast isomerase, together with a new method of analysis involving /sup 14/C and /sup 3/H doubly labeled substrates, have been used to investigate the energetics of the mutant enzyme catalyzed reaction. The deuterium kinetic isotope effects observed with the mutant isomerase using (1(R)-/sup 2/H)dihydroxyacetone phosphate and (2-/sup 2/H)glyceraldehyde 3-phosphate are 2.15 +/- 0.04 and 2.4 +/- 0.1, respectively. These results lead to the conclusion that substitution of Gln for His-95 so impairs the ability of the enzyme to stabilize the reaction intermediate that there is a change in the pathways of proton transfer mediated by the mutant enzyme.

  10. EVALUATION OF THE DORSET SHEEP AS A PREDICTIVE ANIMAL MODEL FOR THE RESPONSE OF GLUCOSE-6-PHOSPHATE DEHYDROGENASE-DEFICIENT HUMAN ERYTHOCYTES TO A PROPOSED SYSTEMIC TOXIC OZONE INTERMEDIATE, METHYL OLEATE OZONIDE

    EPA Science Inventory

    Erythrocytes of both glucose-6-phosphate dehydrogenase (G-6-PD)-deficient humans and Dorest sheep, an animal model with an erythrocyte G-6-PD deficiency, responded in a dose-dependent manner to the oxidant stress of methyl oleate ozonide (MOO) as measured by decreases in G-6-PD a...

  11. 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

  12. One-Pot Biosynthesis of High-Concentration α-Glucose 1-Phosphate from Starch by Sequential Addition of Three Hyperthermophilic Enzymes.

    PubMed

    Zhou, Wei; You, Chun; Ma, Hongwu; Ma, Yanhe; Zhang, Y-H Percival

    2016-03-01

    α-Glucose 1-phosphate (G1P) is synthesized from 5% (w/v) corn starch and 1 M phosphate mediated by α-glucan phosphorylase (αGP) from the thermophilic bacterium Thermotoga maritima at pH 7.2 and 70 °C. To increase G1P yield from corn starch containing branched amylopectin, a hyper-thermostable isoamylase from Sulfolobus tokodaii was added for simultaneous starch gelatinization and starch-debranching hydrolysis at 85 °C and pH 5.5 before αGP use. The pretreatment of isoamylase increased G1P titer from 120 mM to 170 mM. To increase maltose and maltotriose utilization, the third thermostable enzyme, 4-glucanotransferase (4GT) from Thermococcus litoralis, was added during the late stage of G1P biotransformation, further increasing G1P titer to 200 mM. This titer is the highest G1P level obtained on starch or its derived products (maltodextrin and soluble starch). This study suggests that in vitro multienzyme biotransformation has an advantage of great engineering flexibility in terms of space and time compared with microbial fermentation. PMID:26832825

  13. Real-time monitoring of glucose-6-phosphate dehydrogenase activity using liquid droplet arrays and its application to human plasma samples.

    PubMed

    Jung, Se-Hui; Ji, Su-Hyun; Han, Eun-Taek; Park, Won Sun; Hong, Seok-Ho; Kim, Young-Myeong; Ha, Kwon-Soo

    2016-05-15

    Glucose-6-phosphate dehydrogenase (G6PD) regulates nicotinamide adenine dinucleotide phosphate (NADPH) levels and is related to the pathogenesis of various diseases, including G6PD deficiency, type 2 diabetes, aldosterone-induced endothelial dysfunction, and cancer. Therefore, a highly sensitive array-based assay for determining quantitative G6PD activity is required. Here, we developed an on-chip G6PD activity assay using liquid droplet fluorescence arrays. Quantitative G6PD activity was determined by calculating reduced resorufin concentrations in liquid droplets. The limit of detection (LOD) of this assay was 0.162 mU/ml (2.89 pM), which is much more sensitive than previous assays. We used our activity assay to determine kinetic parameters, including Michaelis-Menten constants (Km) and maximum rates of enzymatic reaction (Vmax) for NADP(+) and G6P, and half-maximal inhibitory concentrations (IC50). We successfully applied this new assay to determine G6PD activity in human plasma from normal healthy individuals (n=30) and patients with inflammation (n=30). The inflammatory group showed much higher G6PD activities than did the normal group (p<0.001), with a high area under the curve value of 0.939. Therefore, this new activity assay has the potential to be used for diagnosis of G6PD-associated diseases and utilizing kinetic studies. PMID:26802575

  14. Disruption of the Phosphate Transporter Pit1 in Hepatocytes Improves Glucose Metabolism and Insulin Signaling by Modulating the USP7/IRS1 Interaction.

    PubMed

    Forand, Anne; Koumakis, Eugénie; Rousseau, Alice; Sassier, Yohann; Journe, Clément; Merlin, Jean-François; Leroy, Christine; Boitez, Valérie; Codogno, Patrice; Friedlander, Gérard; Cohen, Isabelle

    2016-09-01

    The liver plays a central role in whole-body lipid and glucose homeostasis. Increasing dietary fat intake results in increased hepatic fat deposition, which is associated with a risk for development of insulin resistance and type 2 diabetes. In this study, we demonstrate a role for the phosphate inorganic transporter 1 (PiT1/SLC20A1) in regulating metabolism. Specific knockout of Pit1 in hepatocytes significantly improved glucose tolerance and insulin sensitivity, enhanced insulin signaling, and decreased hepatic lipogenesis. We identified USP7 as a PiT1 binding partner and demonstrated that Pit1 deletion inhibited USP7/IRS1 dissociation upon insulin stimulation. This prevented IRS1 ubiquitination and its subsequent proteasomal degradation. As a consequence, delayed insulin negative feedback loop and sustained insulin signaling were observed. Moreover, PiT1-deficient mice were protected against high-fat-diet-induced obesity and diabetes. Our findings indicate that PiT1 has potential as a therapeutic target in the context of metabolic syndrome, obesity, and diabetes. PMID:27568561

  15. Glucose Metabolism in Neisseria gonorrhoeae

    PubMed Central

    Morse, Stephen A.; Stein, Stefanie; Hines, James

    1974-01-01

    The metabolism of glucose was examined in several clinical isolates of Neisseria gonorrhoeae. Radiorespirometric studies revealed that growing cells metabolized glucose by a combination on the Entner-Doudoroff and pentose phosphate pathways. A portion of the glyceraldehyde-3-phosphate formed via the Entner-Doudoroff pathway was recycled by conversion to glucose-6-phosphate. Subsequent catabolism of this glucose-6-phosphate by either the Entner-Doudoroff or pentose phosphate pathways yielded CO2 from the original C6 of glucose. Enzyme analyses confirmed the presence of all enzymes of the Entner-Doudoroff, pentose phosphate, and Embden-Meyerhof-Parnas pathways. There was always a high specific activity of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) relative to that of 6-phosphogluconate dehydrogenase (EC 1.1.1.44). The glucose-6-phosphate dehydrogenase utilized either nicotinamide adenine dinucleotide phosphate or nicotinamide adenine dinucleotide as electron acceptor. Acetate was the only detectable nongaseous end product of glucose metabolism. Following the disappearance of glucose, acetate was metabolized by the tricarboxylic acid cycle as evidenced by the preferential oxidation of [1-14C]acetate over that of [2-14C]acetate. When an aerobically grown log-phase culture was subjected to anaerobic conditions, lactate and acetate were formed from glucose. Radiorespirometric studies showed that under these conditions, glucose was dissimilated entirely by the Entner-Doudoroff pathway. Further studies determined that this anaerobic dissimilation of glucose was not growth dependent. PMID:4156358

  16. The oxidative pentose phosphate pathway is the primary source of NADPH for lipid overproduction from glucose in Yarrowia lipolytica.

    PubMed

    Wasylenko, Thomas M; Ahn, Woo Suk; Stephanopoulos, Gregory

    2015-07-01

    Oleaginous microbes represent an attractive means of converting a diverse range of feedstocks into oils that can be transesterified to biodiesel. However, the mechanism of lipid overproduction in these organisms is incompletely understood, hindering the development of strategies for engineering superior biocatalysts for "single-cell oil" production. In particular, it is unclear which pathways are used to generate the large quantities of NADPH required for overproduction of the highly reduced fatty acid species. While early studies implicated malic enzyme as having a key role in production of lipogenic NADPH in oleaginous fungi, several recent reports have cast doubts as to whether malic enzyme may contribute to production of lipogenic NADPH in the model oleaginous yeast Yarrowia lipolytica. To address this problem we have used (13)C-Metabolic Flux Analysis to estimate the metabolic flux distributions during lipid accumulation in two Y. lipolytica strains; a control strain and a previously published engineered strain capable of producing lipids at roughly twice the yield. We observe a dramatic rearrangement of the metabolic flux distribution in the engineered strain which supports lipid overproduction. The NADPH-producing flux through the oxidative Pentose Phosphate Pathway is approximately doubled in the engineered strain in response to the roughly two-fold increase in fatty acid biosynthesis, while the flux through malic enzyme does not differ significantly between the two strains. Moreover, the estimated rate of NADPH production in the oxidative Pentose Phosphate Pathway is in good agreement with the estimated rate of NADPH consumption in fatty acid biosynthesis in both strains. These results suggest the oxidative Pentose Phosphate Pathway is the primary source of lipogenic NADPH in Y. lipolytica. PMID:25747307

  17. Comparison of the xylose reductase-xylitol dehydrogenase and the xylose isomerase pathways for xylose fermentation by recombinant Saccharomyces cerevisiae

    PubMed Central

    Karhumaa, Kaisa; Sanchez, Rosa Garcia; Hahn-Hägerdal, Bärbel; Gorwa-Grauslund, Marie-F

    2007-01-01

    Background Two heterologous pathways have been used to construct recombinant xylose-fermenting Saccharomyces cerevisiae strains: i) the xylose reductase (XR) and xylitol dehydrogenase (XDH) pathway and ii) the xylose isomerase (XI) pathway. In the present study, the Pichia stipitis XR-XDH pathway and the Piromyces XI pathway were compared in an isogenic strain background, using a laboratory host strain with genetic modifications known to improve xylose fermentation (overexpressed xylulokinase, overexpressed non-oxidative pentose phosphate pathway and deletion of the aldose reductase gene GRE3). The two isogenic strains and the industrial xylose-fermenting strain TMB 3400 were studied regarding their xylose fermentation capacity in defined mineral medium and in undetoxified lignocellulosic hydrolysate. Results In defined mineral medium, the xylose consumption rate, the specific ethanol productivity, and the final ethanol concentration were significantly higher in the XR- and XDH-carrying strain, whereas the highest ethanol yield was achieved with the strain carrying XI. While the laboratory strains only fermented a minor fraction of glucose in the undetoxified lignocellulose hydrolysate, the industrial strain TMB 3400 fermented nearly all the sugar available. Xylitol was formed by the XR-XDH-carrying strains only in mineral medium, whereas in lignocellulose hydrolysate no xylitol formation was detected. Conclusion Despite by-product formation, the XR-XDH xylose utilization pathway resulted in faster ethanol production than using the best presently reported XI pathway in the strain background investigated. The need for robust industrial yeast strains for fermentation of undetoxified spruce hydrolysates was also confirmed. PMID:17280608

  18. 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

  19. Structure and Stability of the Dimeric Triosephosphate Isomerase from the Thermophilic Archaeon Thermoplasma acidophilum

    PubMed Central

    Park, Sang Ho; Kim, Hyoun Sook; Park, Mi Seul; Moon, Sojin; Song, Mi Kyung; Park, Han Su; Hahn, Hyunggu; Kim, Soon-Jong; Bae, Euiyoung; Kim, Hyun-Jung; Han, Byung Woo

    2015-01-01

    Thermoplasma acidophilum is a thermophilic archaeon that uses both non-phosphorylative Entner-Doudoroff (ED) pathway and Embden-Meyerhof-Parnas (EMP) pathway for glucose degradation. While triosephosphate isomerase (TPI), a well-known glycolytic enzyme, is not involved in the ED pathway in T. acidophilum, it has been considered to play an important role in the EMP pathway. Here, we report crystal structures of apo- and glycerol-3-phosphate-bound TPI from T. acidophilum (TaTPI). TaTPI adopts the canonical TIM-barrel fold with eight α-helices and parallel eight β-strands. Although TaTPI shares ~30% sequence identity to other TPIs from thermophilic species that adopt tetrameric conformation for enzymatic activity in their harsh physiological environments, TaTPI exists as a dimer in solution. We confirmed the dimeric conformation of TaTPI by analytical ultracentrifugation and size-exclusion chromatography. Helix 5 as well as helix 4 of thermostable tetrameric TPIs have been known to play crucial roles in oligomerization, forming a hydrophobic interface. However, TaTPI contains unique charged-amino acid residues in the helix 5 and adopts dimer conformation. TaTPI exhibits the apparent Td value of 74.6°C and maintains its overall structure with some changes in the secondary structure contents at extremely acidic conditions (pH 1–2). Based on our structural and biophysical analyses of TaTPI, more compact structure of the protomer with reduced length of loops and certain patches on the surface could account for the robust nature of Thermoplasma acidophilum TPI. PMID:26709515

  20. Post-Translational Regulation of the Glucose-6-Phosphatase Complex by Cyclic Adenosine Monophosphate Is a Crucial Determinant of Endogenous Glucose Production and Is Controlled by the Glucose-6-Phosphate Transporter.

    PubMed

    Soty, Maud; Chilloux, Julien; Delalande, François; Zitoun, Carine; Bertile, Fabrice; Mithieux, Gilles; Gautier-Stein, Amandine

    2016-04-01

    The excessive endogenous glucose production (EGP) induced by glucagon participates in the development of type 2 diabetes. To further understand this hormonal control, we studied the short-term regulation by cyclic adenosine monophosphate (cAMP) of the glucose-6-phosphatase (G6Pase) enzyme, which catalyzes the last reaction of EGP. In gluconeogenic cell models, a 1-h treatment by the adenylate cyclase activator forskolin increased G6Pase activity and glucose production independently of any change in enzyme protein amount or G6P content. Using specific inhibitors or protein overexpression, we showed that the stimulation of G6Pase activity involved the protein kinase A (PKA). Results of site-directed mutagenesis, mass spectrometry analyses, and in vitro phosphorylation experiments suggested that the PKA stimulation of G6Pase activity did not depend on a direct phosphorylation of the enzyme. However, the temperature-dependent induction of both G6Pase activity and glucose release suggested a membrane-based mechanism. G6Pase is composed of a G6P transporter (G6PT) and a catalytic unit (G6PC). Surprisingly, we demonstrated that the increase in G6PT activity was required for the stimulation of G6Pase activity by forskolin. Our data demonstrate the existence of a post-translational mechanism that regulates G6Pase activity and reveal the key role of G6PT in the hormonal regulation of G6Pase activity and of EGP. PMID:26958868

  1. 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

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

    PubMed

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

    2015-12-01

    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

  3. Production and Purification of Extracellular D-Xylose Isomerase from an Alkaliphilic, Thermophilic Bacillus sp. †

    PubMed Central

    Chauthaiwale, Jyoti; Rao, Mala

    1994-01-01

    An alkaliphilic, thermophilic Bacillus sp. (NCIM 59) produced extracellular xylose isomerase at pH 10 and 50°C by using xylose or wheat bran as the carbon source. The distribution of xylose isomerase as a function of growth in comparison with distributions of extra- and intracellular marker enzymes such as xylanase and β-galactosidase revealed that xylose isomerase was truly secreted as an extracellular enzyme and was not released because of sporulation or lysis. The enzyme was purified to homogeneity by ammonium sulfate precipitation followed by gel filtration, preparative polyacrylamide gel electrophoresis, and ion-exchange chromatography. The molecular weight of xylose isomerase was estimated to be 160,000 by gel filtration and 50,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating the presence of three subunits. The enzyme is most active at pH 8.0 and with incubation at 85°C for 20 min. Divalent metal ions Mg2+, Co2+, and Mn2+ were required for maximum activity of the enzyme. The Km values for D-xylose and D-glucose at 80°C and pH 7.5 were 6.66 and 142 mM, respectively, while Kcat values were 2.3 × 102 s-1 and 0.5 × 102 s-1, respectively. Images PMID:16349464

  4. Glycolysis and the pentose phosphate pathway after human traumatic brain injury: microdialysis studies using 1,2-13C2 glucose

    PubMed Central

    Jalloh, Ibrahim; Carpenter, Keri L H; Grice, Peter; Howe, Duncan J; Mason, Andrew; Gallagher, Clare N; Helmy, Adel; Murphy, Michael P; Menon, David K; Carpenter, T Adrian; Pickard, John D; Hutchinson, Peter J

    2015-01-01

    Increased ‘anaerobic' glucose metabolism is observed after traumatic brain injury (TBI) attributed to increased glycolysis. An alternative route is the pentose phosphate pathway (PPP), which generates putatively protective and reparative molecules. To compare pathways we employed microdialysis to perfuse 1,2-13C2 glucose into the brains of 15 TBI patients and macroscopically normal brain in six patients undergoing surgery for benign tumors, and to simultaneously collect products for nuclear magnetic resonance (NMR) analysis. 13C enrichment for glycolytic 2,3-13C2 lactate was the median 5.4% (interquartile range (IQR) 4.6–7.5%) in TBI brain and 4.2% (2.4–4.4%) in ‘normal' brain (P<0.01). The ratio of PPP-derived 3-13C lactate to glycolytic 2,3-13C2 lactate was median 4.9% (3.6–8.2%) in TBI brain and 6.7% (6.3–8.9%) in ‘normal' brain. An inverse relationship was seen for PPP-glycolytic lactate ratio versus PbtO2 (r=−0.5, P=0.04) in TBI brain. Thus, glycolytic lactate production was significantly greater in TBI than ‘normal' brain. Several TBI patients exhibited PPP–lactate elevation above the ‘normal' range. There was proportionally greater PPP-derived lactate production with decreasing PbtO2. The study raises questions about the roles of the PPP and glycolysis after TBI, and whether they can be manipulated to achieve a better outcome. This study is the first direct comparison of glycolysis and PPP in human brain. PMID:25335801

  5. Proteomic and biochemical basis for enhanced growth yield of Enterobacter sp. LCR1 on insoluble phosphate medium.

    PubMed

    Kumar, Arvind; Rai, Lal Chand

    2015-01-01

    Proteomics and biochemical analyses were used to unravel the basis for higher growth yield of Enterobacter sp. LCR1 on insoluble phosphate medium compared to soluble. Proteomic analysis using 2-DE, MALDI-TOF/MS and LC-MS revealed the involvement of nine proteins. Down-regulation of fructose bisphosphate aldolase with decreased concentrations of glucose-6-phosphate and fructose-6-phosphate indicated diminished glycolysis. However, up-regulation of phosphoglycerate mutase, increase in the activities of 6-phosphogluconate dehydratase, 2-keto-3-deoxy-6-phosphogluconate aldolase and 6-phosphogluconate dehydrogenase suggested induction of Entner-Doudoroff and pentose phosphate pathways. These pathways generate sufficient energy from gluconic acid, which is also used for biosynthesis as indicated by up-regulation of elongation factor Tu, elongation factor G and protein disulfide isomerase. Increased reactive oxygen species (ROS) formation resulting from organic acid oxidation leads to overexpressed manganese superoxide dismutase and increased activities of catalase and ascorbate peroxidase. Thus the organism uses gluconate instead of glucose for energy, while alleviating extra ROS formation by oxidative defense enzymes. PMID:25053519

  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. Efficient production of a thermophilic 2-deoxyribose-5-phosphate aldolase in glucose-limited fed-batch cultivations of Escherichia coli by continuous lactose induction strategy.

    PubMed

    Pei, Xiao-Lin; Wang, Qiu-Yan; Li, Cheng-Lu; Qiu, Xiao-Feng; Xie, Kai-Lin; Huang, Li-Feng; Wang, An-Ming; Zeng, Zhao-Wu; Xie, Tian

    2011-09-01

    The production of a thermophilic 2-deoxyribose-5-phosphate aldolases (DERA) in Escherichia coli BL21 under continuous lactose induction strategy was investigated. The process was combined with the exponential feeding method, controlling the feeding rate to maintain the specific growth rate at 0.15 h(-1). The results indicate that the lactose concentration in the feed medium affected directly the expression of the target protein. The use of 50 g/L in the feed medium resulted in the biomass concentration of 39.3 g DCW/L, and an expression level of above 30%, and the maximum final DERA concentration of 16,200 U/L. Furthermore, the acetate concentration remained at a low level in the fed-batch phase, less than 0.5 g/L. In conclusion, combining glucose feeding with lactose induction is a more powerful way to achieve high cell density cultures and to efficiently produce the thermophilic DERA. The results also indicate the potential industrial utility in the scale production of other recombinant proteins. PMID:21509600

  8. 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

  9. 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

  10. Red blood cell indices and prevalence of hemoglobinopathies and glucose 6 phosphate dehydrogenase deficiencies in male Tanzanian residents of Dar es Salaam.

    PubMed

    Mwakasungula, Solomon; Schindler, Tobias; Jongo, Said; Moreno, Elena; Kamaka, Kasimu; Mohammed, Mgeni; Joseph, Selina; Rashid, Ramla; Athuman, Thabit; Tumbo, Anneth Mwasi; Hamad, Ali; Lweno, Omar; Tanner, Marcel; Shekalaghe, Seif; Daubenberger, Claudia A

    2014-01-01

    Hemoglobinopathies, disorders of hemoglobin structure and production, are one of the most common monogenic disorders in humans. Glucose 6 phosphate dehydrogenase deficiency (G6PD) is an inherited enzymopathy resulting in increased oxygen stress susceptibility of red blood cells. The distributions of these genetic traits in populations living in tropical and subtropical regions where malaria has been or is still present are thought to result from survival advantage against severe life threatening malaria disease. 384 male Tanzanian volunteers residing in Dar es Salaam were typed for G6PD, sickle cell disease and α-thalassemia. The most prominent red blood cell polymorphism was heterozygous α(+)-thalassemia (37.8%), followed by the G6PD(A) deficiency (16.4%), heterozygous sickle cell trait (15.9%), G6PD(A-) deficiency (13.5%) and homozygous α(+)-thalassemia (5.2%). 35%, 45%, 17% and 3% of these volunteers were carriers of wild type gene loci, one, two or three of these hemoglobinopathies, respectively. We find that using a cut off value of 28.6 pg. for mean corpuscular hemoglobin (MCH), heterozygous α(+)-thalassemia can be predicted with a sensitivity of 84% and specificity of 72% in this male population. All subjects carrying homozygous α(+)-thalassemia were identified based on their MCH value < 28.6 pg. PMID:25755846

  11. 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

  12. Somatic-cell selection is a major determinant of the blood-cell phenotype in heterozygotes for glucose-6-phosphate dehydrogenase mutations causing severe enzyme deficiency.

    PubMed Central

    Filosa, S.; Giacometti, N.; Wangwei, C.; De Mattia, D.; Pagnini, D.; Alfinito, F.; Schettini, F.; Luzzatto, L.; Martini, G.

    1996-01-01

    X-chromosome inactivation in mammals is regarded as an essentially random process, but the resulting somatic-cell mosaicism creates the opportunity for cell selection. In most people with red-blood-cell glucose-6-phosphate dehydrogenase (G6PD) deficiency, the enzyme-deficient phenotype is only moderately expressed in nucleated cells. However, in a small subset of hemizygous males who suffer from chronic nonspherocytic hemolytic anemia, the underlying mutations (designated class I) cause more-severe G6PD deficiency, and this might provide an opportunity for selection in heterozygous females during development. In order to test this possibility we have analyzed four heterozygotes for class I G6PD mutations: two with G6PD Portici (1178G-->A) and two with G6PD Bari (1187C-->T). We found that in fractionated blood cell types (including erythroid, myeloid, and lymphoid cell lineages) there was a significant excess of G6PD-normal cells. The significant concordance that we have observed in the degree of imbalance in the different blood-cell lineages indicates that a selective mechanism is likely to operate at the level of pluripotent blood stem cells. Thus, it appears that severe G6PD deficiency affects adversely the proliferation or the survival of nucleated blood cells and that this phenotypic characteristic is critical during hematopoiesis. Images Figure 1 Figure 3 Figure 4 Figure 5 Figure 6 PMID:8808605

  13. An In vivo Drug Screening Model Using Glucose-6-Phosphate Dehydrogenase Deficient Mice to Predict the Hemolytic Toxicity of 8-Aminoquinolines

    PubMed Central

    Zhang, Peng; Gao, Xiugong; Ishida, Hiroshi; Amnuaysirikul, Jack; Weina, Peter J.; Grogl, Max; O'Neil, Michael T.; Li, Qigui; Caridha, Diana; Ohrt, Colin; Hickman, Mark; Magill, Alan J.; Ray, Prabhati

    2013-01-01

    Anti-malarial 8-aminoquinolines drugs cause acute hemolytic anemia in individuals with glucose-6-phosphate dehydrogenase deficiency (G6PDD). Efforts to develop non-hemolytic 8-aminoquinolines have been severely limited caused by the lack of a predictive in vivo animal model of hemolytic potential that would allow screening of candidate compounds. This report describes a G6PDD mouse model with a phenotype closely resembling the G6PDD phenotype found in the African A-type G6PDD human. These G6PDD mice, given different doses of primaquine, which used as a reference hemolytic drug, display a full array of hemolytic anemia parameters, consistently and reproducibly. The hemolytic and therapeutic indexes were generated for evaluation of hemotoxicity of drugs. This model demonstrated a complete hemolytic toxicity response to another known hemolytic antimalarial drug, pamaquine, but no response to non-hemolytic drugs, chloroquine and mefloquine. These results suggest that this model is suitable for evaluation of selected 8-AQ type candidate antimalarial drugs for their hemolytic potential. PMID:23530079

  14. Two new mutations of the glucose-6-phosphate dehydrogenase (G6PD) gene associated with haemolytic anaemia: clinical, biochemical and molecular relationships.

    PubMed

    Zarza, R; Pujades, A; Rovira, A; Saavedra, R; Fernandez, J; Aymerich, M; Vives Corrons, J L

    1997-09-01

    In two unrelated Spanish males with glucose-6-phosphate dehydrogenase (G6PD) deficiency and haemolytic anaemia, and two different novel point mutations in the G6PD gene, have been identified. A C to T transition at nucleotide 406 resulting in a (136) Arg to Cys substitution and a C to G transition at nucleotide 1155 resulting in a (385) Cys to Trp substitution. These two molecular defects have not been described before and are designated G6PD Valladolid 406 C-->T and G6PD Madrid 1155 C-->G. In vitro biochemical characterization of both mutant enzymes showed important differences in their molecular properties according to their different clinical behaviour. In G6PD Valladolid, the mutation of which is located in exon 5, the normal in vitro heat stability may explain its mild clinical expression (low-grade haemolysis interrupted by an acute haemolytic crisis at age 70). In G6PD Madrid, the mutation, located in exon 10, results in a deficient variant associated with neonatal jaundice and life-long chronic nonspherocytic haemolytic anaemia (CNSHA). This finding further emphasizes the importance of this specific region of the G6PD gene in the stabilization of the G6PD molecule. Putative relationships between these single point mutations and the molecular properties of the mutant enzymes are also discussed. PMID:9332310

  15. 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

  16. 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

  17. Evolutionary engineering of a glycerol-3-phosphate dehydrogenase-negative, acetate-reducing Saccharomyces cerevisiae strain enables anaerobic growth at high glucose concentrations

    PubMed Central

    Guadalupe-Medina, Víctor; Metz, Benjamin; Oud, Bart; van Der Graaf, Charlotte M; Mans, Robert; Pronk, Jack T; van Maris, Antonius J A

    2014-01-01

    Glycerol production by Saccharomyces cerevisiae, which is required for redox-cofactor balancing in anaerobic cultures, causes yield reduction in industrial bioethanol production. Recently, glycerol formation in anaerobic S. cerevisiae cultures was eliminated by expressing Escherichia coli (acetylating) acetaldehyde dehydrogenase (encoded by mhpF) and simultaneously deleting the GPD1 and GPD2 genes encoding glycerol-3-phosphate dehydrogenase, thus coupling NADH reoxidation to reduction of acetate to ethanol. Gpd– strains are, however, sensitive to high sugar concentrations, which complicates industrial implementation of this metabolic engineering concept. In this study, laboratory evolution was used to improve osmotolerance of a Gpd– mhpF-expressing S. cerevisiae strain. Serial batch cultivation at increasing osmotic pressure enabled isolation of an evolved strain that grew anaerobically at 1 M glucose, at a specific growth rate of 0.12 h−1. The evolved strain produced glycerol at low concentrations (0.64 ± 0.33 g l−1). However, these glycerol concentrations were below 10% of those observed with a Gpd+ reference strain. Consequently, the ethanol yield on sugar increased from 79% of the theoretical maximum in the reference strain to 92% for the evolved strains. Genetic analysis indicated that osmotolerance under aerobic conditions required a single dominant chromosomal mutation, and one further mutation in the plasmid-borne mhpF gene for anaerobic growth. PMID:24004455

  18. Glucose-6-Phosphate Dehydrogenase Deficiency in an Endemic Area for Malaria in Manaus: A Cross-Sectional Survey in the Brazilian Amazon

    PubMed Central

    Santana, Marli Stela; de Lacerda, Marcus Vinícius Guimarães; Barbosa, Maria das Graças Vale; Alecrim, Wilson Duarte; Alecrim, Maria das Graças Costa

    2009-01-01

    Background There is a paucity of information regarding glucose-6-phosphate dehydrogenase (G6PD) deficiency in endemic areas for malaria in Latin America. Methodology/Principal Findings This study determined the prevalence of the G6PD deficiency in 200 male non-consanguineous individuals residing in the Ismail Aziz Community, on the outskirts of Manaus (Brazilian Amazon). Six individuals (3%) were deficient using the qualitative Brewer's test. Gel electrophoresis showed that five of these patients were G6PD A−. The deficiency was not associated with the ethnic origin (P = 0.571). In a multivariate logistic regression analysis, G6PD deficiency protected against three or more episodes of malaria (P = 0.049), independently of the age, and was associated with a history of jaundice (P = 0.020) and need of blood transfusion (P = 0.045) during previous treatment for malarial infection, independently of the age and the previous malarial exposure. Conclusions/Significance The frequency of G6PD deficiency was similar to other studies performed in Brazil and the finding of a predominant G6PD A− variant will help the clinical management of patients with drug-induced haemolysis. The history of jaundice and blood transfusion during previous malarial infection may trigger the screening of patients for G6PD deficiency. The apparent protection against multiple malarial infections in an area primarily endemic for Plasmodium vivax needs further investigation. PMID:19370159

  19. Isocitrate Dehydrogenase Is Important for Nitrosative Stress Resistance in Cryptococcus neoformans, but Oxidative Stress Resistance Is Not Dependent on Glucose-6-Phosphate Dehydrogenase▿

    PubMed Central

    Brown, Sarah M.; Upadhya, Rajendra; Shoemaker, James D.; Lodge, Jennifer K.

    2010-01-01

    The opportunistic intracellular fungal pathogen Cryptococcus neoformans depends on many antioxidant and denitrosylating proteins and pathways for virulence in the immunocompromised host. These include the glutathione and thioredoxin pathways, thiol peroxidase, cytochrome c peroxidase, and flavohemoglobin denitrosylase. All of these ultimately depend on NADPH for either catalytic activity or maintenance of a reduced, functional form. The need for NADPH during oxidative stress is well established in many systems, but a role in resistance to nitrosative stress has not been as well characterized. In this study we investigated the roles of two sources of NADPH, glucose-6-phosphate dehydrogenase (Zwf1) and NADP+-dependent isocitrate dehydrogenase (Idp1), in production of NADPH and resistance to oxidative and nitrosative stress. Deletion of ZWF1 in C. neoformans did not result in an oxidative stress sensitivity phenotype or changes in the amount of NADPH produced during oxidative stress compared to those for the wild type. Deletion of IDP1 resulted in greater sensitivity to nitrosative stress than to oxidative stress. The amount of NADPH increased 2-fold over that in the wild type during nitrosative stress, and yet the idp1Δ strain accumulated more mitochondrial damage than the wild type during nitrosative stress. This is the first report of the importance of Idp1 and NADPH for nitrosative stress resistance. PMID:20400467

  20. Red blood cell indices and prevalence of hemoglobinopathies and glucose 6 phosphate dehydrogenase deficiencies in male Tanzanian residents of Dar es Salaam

    PubMed Central

    Mwakasungula, Solomon; Schindler, Tobias; Jongo, Said; Moreno, Elena; Kamaka, Kasimu; Mohammed, Mgeni; Joseph, Selina; Rashid, Ramla; Athuman, Thabit; Tumbo, Anneth Mwasi; Hamad, Ali; Lweno, Omar; Tanner, Marcel; Shekalaghe, Seif; Daubenberger, Claudia A

    2014-01-01

    Hemoglobinopathies, disorders of hemoglobin structure and production, are one of the most common monogenic disorders in humans. Glucose 6 phosphate dehydrogenase deficiency (G6PD) is an inherited enzymopathy resulting in increased oxygen stress susceptibility of red blood cells. The distributions of these genetic traits in populations living in tropical and subtropical regions where malaria has been or is still present are thought to result from survival advantage against severe life threatening malaria disease. 384 male Tanzanian volunteers residing in Dar es Salaam were typed for G6PD, sickle cell disease and α-thalassemia. The most prominent red blood cell polymorphism was heterozygous α+-thalassemia (37.8%), followed by the G6PD(A) deficiency (16.4%), heterozygous sickle cell trait (15.9%), G6PD(A-) deficiency (13.5%) and homozygous α+-thalassemia (5.2%). 35%, 45%, 17% and 3% of these volunteers were carriers of wild type gene loci, one, two or three of these hemoglobinopathies, respectively. We find that using a cut off value of 28.6 pg. for mean corpuscular hemoglobin (MCH), heterozygous α+-thalassemia can be predicted with a sensitivity of 84% and specificity of 72% in this male population. All subjects carrying homozygous α+-thalassemia were identified based on their MCH value < 28.6 pg. PMID:25755846

  1. 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

  2. Somatic-cell selection is a major determinant of the blood-cell phenotype in heterozygotes for glucose-6-phosphate dehydrogenase mutations causing severe enzyme deficiency

    SciTech Connect

    Filosa, S.; Giacometti, N.; Wangwei, C.; Martini, G.

    1996-10-01

    X-chromosome inactivation in mammals is regarded as an essentially random process, but the resulting somatic-cell mosaicism creates the opportunity for cell selection. In most people with red-blood-cell glucose-6-phosphate dehydrogenase (G6PD) deficiency, the enzyme-deficient phenotype is only moderately expressed in nucleated cells. However, in a small subset of hemizygous males who suffer from chronic nonspherocytic hemolytic anemia, the underlying mutations (designated class I) cause more-severe G6PD deficiency, and this might provide an opportunity for selection in heterozygous females during development. In order to test this possibility we have analyzed four heterozygotes for class I G6PD mutations: two with G6PD Portici (1178G{r_arrow}A) and two with G6PD Bari (1187C{r_arrow}T). We found that in fractionated blood cell types (including erythroid, myeloid, and lymphoid cell lineages) there was a significant excess of G6PD-normal cells. The significant concordance that we have observed in the degree of imbalance in the different blood-cell lineages indicates that a selective mechanism is likely to operate at the level of pluripotent blood stem cells. Thus, it appears that severe G6PD deficiency affects adversely the proliferation or the survival of nucleated blood cells and that this phenotypic characteristic is critical during hematopoiesis. 65 refs., 6 figs., 3 tabs.

  3. 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.

  4. 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

    2015-01-01

    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

  5. Dihydroxyacetone production in an engineered Escherichia coli through expression of Corynebacterium glutamicum dihydroxyacetone phosphate dephosphorylase.

    PubMed

    Jain, Vishist Kumar; Tear, Crystal Jing Ying; Lim, Chan Yuen

    2016-05-01

    Dihydroxyacetone (DHA) has several industrial applications such as a tanning agent in tanning lotions in the cosmetic industry; its production via microbial fermentation would present a more sustainable option for the future. Here we genetically engineered Escherichia coli (E. coli) for DHA production from glucose. Deletion of E. coli triose phosphate isomerase (tpiA) gene was carried out to accumulate dihydroxyacetone phosphate (DHAP), for use as the main intermediate or precursor for DHA production. The accumulated DHAP was then converted to DHA through the heterologous expression of Corynebacterium glutamicum DHAP dephosphorylase (cghdpA) gene. To conserve DHAP exclusively for DHA production we removed methylglyoxal synthase (mgsA) gene in the ΔtpiA strain. This drastically improved DHA production from 0.83g/l (0.06g DHA/g glucose) in the ΔtpiA strain bearing cghdpA to 5.84g/l (0.41g DHA/g glucose) in the ΔtpiAΔmgsA double mutant containing the same gene. To limit the conversion of intracellular DHA to glycerol, glycerol dehydrogenase (gldA) gene was further knocked out resulting in a ΔtpiAΔmgsAΔgldA triple mutant. This triple mutant expressing the cghdpA gene produced 6.60g/l of DHA at 87% of the maximum theoretical yield. In summary, we demonstrated an efficient system for DHA production in genetically engineered E. coli strain. PMID:26992791

  6. 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

  7. Biosynthesis of dTDP-3-acetamido-3,6-dideoxy-alpha-D-glucose.

    PubMed

    Pföstl, Andreas; Zayni, Sonja; Hofinger, Andreas; Kosma, Paul; Schäffer, Christina; Messner, Paul

    2008-02-15

    Derivatives of 3-amino-3,6-dideoxyhexoses are widespread in Nature. They are part of the repeating units of lipopolysaccharide O-antigens, of the glycan moiety of S-layer (bacterial cell surface layer) glycoproteins and also of many antibiotics. In the present study, we focused on the elucidation of the biosynthesis pathway of dTDP-alpha-D-Quip3NAc (dTDP-3-acetamido-3,6-dideoxy-alpha-D-glucose) from the Gram-positive, anaerobic, thermophilic organism Thermoanaerobacterium thermosaccharolyticum E207-71, which carries Quip3NAc in its S-layer glycan. The biosynthesis of dTDP-alpha-D-Quip3NAc involves five enzymes, namely a transferase, a dehydratase, an isomerase, a transaminase and a transacetylase, and follows a pathway similar to that of dTDP-alpha-D-Fucp3NAc (dTDP-3-acetamido-3,6-dideoxy-alpha-D-galactose) biosynthesis in Aneurinibacillus thermoaerophilus L420-91(T). The ORFs (open reading frames) of interest were cloned, overexpressed in Escherichia coli and purified. To elucidate the enzymatic cascade, the different products were purified by HPLC and characterized by NMR spectroscopy. The initiating reactions catalysed by the glucose-1-phosphate thymidylyltransferase RmlA and the dTDP-D-glucose-4,6-dehydratase RmlB are well established. The subsequent isomerase was shown to be capable of forming a dTDP-3-oxo-6-deoxy-D-glucose intermediate from the RmlB product dTDP-4-oxo-6-deoxy-D-glucose, whereas the isomerase involved in the dTDP-alpha-D-Fucp3NAc pathway synthesizes dTDP-3-oxo-6-deoxy-D-galactose. The subsequent reaction steps of either pathway involve a transaminase and a transacetylase, leading to the specific production of nucleotide-activated 3-acetamido-3,6-dideoxy-alpha-D-glucose and 3-acetamido-3,6-dideoxy-alpha-D-galactose respectively. Sequence comparison of the ORFs responsible for the biosynthesis of dTDP-alpha-D-Quip3NAc revealed homologues in Gram-negative as well as in antibiotic-producing Gram-positive bacteria. There is strong evidence that the

  8. Biosynthesis of dTDP-3-acetamido-3,6-dideoxy-α-d-glucose

    PubMed Central

    Pföstl, Andreas; Zayni, Sonja; Hofinger, Andreas; Kosma, Paul; Schäffer, Christina; Messner, Paul

    2015-01-01

    Derivatives of 3-amino-3,6-dideoxyhexoses are widespread in Nature. They are part of the repeating units of lipopolysaccharide O-antigens, of the glycan moiety of S-layer (bacterial cell surface layer) glycoproteins and also of many antibiotics. In the present study, we focused on the elucidation of the biosynthesis pathway of dTDP-α-d-Quip3NAc (dTDP-3-acetamido-3,6-dideoxy-α-d-glucose) from the Gram-positive, anaerobic, thermophilic organism Thermoanaerobacterium thermosaccharolyticum E207-71, which carries Quip3NAc in its S-layer glycan. The biosynthesis of dTDP-α-d-Quip3NAc involves five enzymes, namely a transferase, a dehydratase, an isomerase, a transaminase and a transacetylase, and follows a pathway similar to that of dTDP-α-d-Fucp3NAc (dTDP-3-acetamido-3,6-dideoxy-α-d-galactose) biosynthesis in Aneurinibacillus thermoaerophilus L420-91T. The ORFs (open reading frames) of interest were cloned, overexpressed in Escherichia coli and purified. To elucidate the enzymatic cascade, the different products were purified by HPLC and characterized by NMR spectroscopy. The initiating reactions catalysed by the glucose-1-phosphate thymidylyltransferase RmlA and the dTDP-d-glucose-4,6-dehydratase RmlB are well established. The subsequent isomerase was shown to be capable of forming a dTDP-3-oxo-6-deoxy-d-glucose intermediate from the RmlB product dTDP-4-oxo-6-deoxy-d-glucose, whereas the isomerase involved in the dTDP-α-d-Fucp3NAc pathway synthesizes dTDP-3-oxo-6-deoxy-d-galactose. The subsequent reaction steps of either pathway involve a transaminase and a transacetylase, leading to the specific production of nucleotide-activated 3-acetamido-3,6-dideoxy-α-d-glucose and 3-acetamido-3,6-dideoxy-α-d-galactose respectively. Sequence comparison of the ORFs responsible for the biosynthesis of dTDP-α-d-Quip3NAc revealed homologues in Gram-negative as well as in antibiotic-producing Gram-positive bacteria. There is strong evidence that the elucidated biosynthesis

  9. 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. PMID:23404100

  10. Generation of C3- and C2-deuterated L-lactic acid by human erythrocytes exposed to D-[1-13C]glucose, D-[2-13C]glucose and D-[6-13C]glucose in the presence of D2O.

    PubMed

    Malaisse, W J; Biesemans, M; Willem, R

    1994-05-01

    1. The generation of C2- and C3-deuterated L-lactate was monitored by 13C NMR in human erythrocytes exposed to D-[1-13C]glucose, D-[2-13C]glucose or D-[6-13C]glucose and incubated in a medium prepared in D2O. 2. The results suggested that the deuteration of the C1 of D-fructose 6-phosphate in the phosphoglucoisomerase reaction, the deuteration of the C1 of D-glyceraldehyde-3-phosphate in the sequence of reactions catalyzed by triose phosphate isomerase and aldolase and the deuteration of the C3 of pyruvate in the reaction catalyzed by pyruvate kinase were all lower than expected from equilibration with D2O. 3. Moreover, about 40% of the molecules of pyruvate generated by glycolysis apparently underwent deuteration on their C3 during interconversion of the 2-keto acid and L-alanine in the reaction catalyzed by glutamate-pyruvate transaminase. 4. The occurrence of the latter process was also documented in cells exposed to exogenous [3-13C]pyruvate. 5. This methodological approach is proposed to provide a new tool to assess in intact cells the extent of back-and-forth interconversion of selected metabolic intermediates. PMID:8005354

  11. Structural and functional characterization of Mycobacterium tuberculosis triosephosphate isomerase

    SciTech Connect

    Connor, Sean E.; Capodagli, Glenn C.; Deaton, Michelle K.; Pegan, Scott D.

    2012-04-18

    Tuberculosis (TB) is a major infectious disease that accounts for over 1.7 million deaths every year. Mycobacterium tuberculosis, the causative agent of tuberculosis, enters the human host by the inhalation of infectious aerosols. Additionally, one third of the world's population is likely to be infected with latent TB. The incidence of TB is on the rise owing in part to the emergence of multidrug-resistant strains. As a result, there is a growing need to focus on novel M. tuberculosis enzyme targets. M. tuberculosis triosephosphate isomerase (MtTPI) is an essential enzyme for gluconeogenetic pathways, making it a potential target for future therapeutics. In order to determine its structure, the X-ray crystal structure of MtTPI has been determined, as well as that of MtTPI bound with a reaction-intermediate analog. As a result, two forms of the active site were revealed. In conjunction with the kinetic parameters obtained for the MtTPI-facilitated conversion of dihydroxyacetone phosphate (DHAP) to D-glyceraldehyde-3-phosphate (D-GAP), this provides a greater structural and biochemical understanding of this enzyme. Additionally, isothermal titration calorimetry was used to determine the binding constant for a reaction-intermediate analog bound to the active site of MtTPI.

  12. Expression, purification, crystallization and preliminary X-ray diffraction analysis of Bifidobacterium adolescentis xylose isomerase

    PubMed Central

    dos Reis, Caio Vinicius; Bernardes, Amanda; Polikarpov, Igor

    2013-01-01

    Xylose isomerase (EC 5.3.1.5) is a key enzyme in xylose metabolism which is industrially important for the transformation of glucose and xylose into fructose and xylulose, respectively. The Bifidobacterium adolescentis xylA gene (NC_008618.1) encoding xylose isomerase (XI) was cloned and the enzyme was overexpressed in Escherichia coli. Purified recombinant XI was crystallized using the sitting-drop vapour-diffusion method with polyethylene glycol 3350 as the precipitating agent. A complete native data set was collected to 1.7 Å resolution using a synchrotron-radiation source. The crystals belonged to the orthorhombic space group P21212, with unit-cell parameters a = 88.78, b = 123.98, c = 78.63 Å. PMID:23695585

  13. Identification of 2-Fluoro-2-deoxy- D-glucose Metabolites by 19F{ 1H} Hetero-RELAY

    NASA Astrophysics Data System (ADS)

    O'Connell, Thomas M.; London, Robert E.

    1995-12-01

    It has been proposed that in mammalian systems the glucose analog 2-fluoro-2-deoxy-D-glucose (FDG) is phosphoryated and subsequently converted to the corresponding mannose derivative via the action of phosphoglucose isomerase. As is generally true in metabolic studies of fluorinated molecules, the fluorine spectrum alone is suggestive, without providing definitive structural evidence, while the use of1H NMR techniques generally suffers from a lack of adequate selectivity. A1H-19F version of the hetero-RELAY experiment has been applied to this problem. Formation of the corresponding C-6 phosphorylated 2-FDG analog with hexokinase, followed by treatment of the resulting phosphorylated products with phosphoglucose isomerase, resulted in the observation of additional19F resonances consistent with the corresponding 2-fluoro-2-deoxy-D-mannose-6-phosphate metabolite. A more definitive product identification was obtained using the hetero-RELAY experiment, which provides a complete19F-decoupled proton spectrum for each of the fluorinated species.

  14. 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. PMID:26009793

  15. 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

  16. Process Integration for the Disruption of Candida guilliermondii Cultivated in Rice Straw Hydrolysate and Recovery of Glucose-6-Phosphate Dehydrogenase by Aqueous Two-Phase Systems.

    PubMed

    Gurpilhares, Daniela B; Pessoa, Adalberto; Roberto, Inês C

    2015-07-01

    Remaining cells of Candida guilliermondii cultivated in hemicellulose-based fermentation medium were used as intracellular protein source. Recovery of glucose-6-phosphate dehydrogenase (G6PD) was attained in conventional aqueous two-phase systems (ATPS) was compared with integrated process involving mechanical disruption of cells followed by ATPS. Influences of polyethylene glycol molar mass (M PEG) and tie line lengths (TLL) on purification factor (PF), yields in top (Y T ) and bottom (Y B ) phases and partition coefficient (K) were evaluated. First scheme resulted in 65.9 % enzyme yield and PF of 2.16 in salt-enriched phase with clarified homogenate (M PEG 1500 g mol(-1), TLL 40 %); Y B of 75.2 % and PF B of 2.9 with unclarified homogenate (M PEG 1000 g mol(-1), TLL 35 %). The highest PF value of integrated process was 2.26 in bottom phase (M PEG 1500 g mol(-1), TLL 40 %). In order to optimize this response, a quadratic model was predicted for the response PFB for process integration. Maximum response achieved was PFB = 3.3 (M PEG 1500 g mol(-1), TLL 40 %). Enzyme characterization showed G6P Michaelis-Menten constant (K M ) equal 0.07-0.05, NADP(+) K M 0.02-1.98 and optimum temperature 70 °C, before and after recovery. Overall, our data confirmed feasibility of disruption/extraction integration for single-step purification of intracellular proteins from remaining yeast cells. PMID:25987135

  17. 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

  18. Cytological mapping of the human glucose-6-phosphate dehydrogenase gene distal to the fragile-X site suggests a high rate of meiotic recombination across this site.

    PubMed

    Szabo, P; Purrello, M; Rocchi, M; Archidiacono, N; Alhadeff, B; Filippi, G; Toniolo, D; Martini, G; Luzzatto, L; Siniscalco, M

    1984-12-01

    The human gene for glucose-6-phosphate dehydrogenase (G6PD) has been subregionally mapped to band Xq28 by segregation analysis in rodent-human somatic cell hybrids [Pai, G. S., Sprinkel, J. A., Do, T. T., Mareni, C. E. & Migeon, B. R. (1980) Proc. Natl. Acad. Sci. USA 77, 2810-2813]. We have previously reported a common type of X-linked mental retardation associated with an inducible fragile site at Xq27-Xq28 segregates in a close linkage relationship with a G6PD variant, but the relative position of G6PD with respect to the fragile site has not yet been established. This fragile-X syndrome has been shown to be closely linked also to a Taq I restriction fragment length polymorphism detected by a cDNA probe for factor IX, and the latter locus has been mapped to the subtelomeric region Xq26-Xq28 [Camerino, G., Mattei, M. G., Mattei, G. F., Jaye, B. & Mandel, J. L. (1983) Nature (London) 306, 701-704]. The in situ hybridization studies reported here provide strong evidence that G6PD is located on the Xq telomeric fragment distal to the fragile site. These observations and the well-established knowledge that the genes for Deutan and Protan colorblindness are closely linked to G6PD, but segregate independently of factor IX deficiency, suggest that the fragile site associated with this type of X-linked mental retardation occurs in a region prone to high frequency of meiotic recombination. PMID:6595664

  19. Cytological mapping of the human glucose-6-phosphate dehydrogenase gene distal to the fragile-X site suggests a high rate of meiotic recombination across this site.

    PubMed Central

    Szabo, P; Purrello, M; Rocchi, M; Archidiacono, N; Alhadeff, B; Filippi, G; Toniolo, D; Martini, G; Luzzatto, L; Siniscalco, M

    1984-01-01

    The human gene for glucose-6-phosphate dehydrogenase (G6PD) has been subregionally mapped to band Xq28 by segregation analysis in rodent-human somatic cell hybrids [Pai, G. S., Sprinkel, J. A., Do, T. T., Mareni, C. E. & Migeon, B. R. (1980) Proc. Natl. Acad. Sci. USA 77, 2810-2813]. We have previously reported a common type of X-linked mental retardation associated with an inducible fragile site at Xq27-Xq28 segregates in a close linkage relationship with a G6PD variant, but the relative position of G6PD with respect to the fragile site has not yet been established. This fragile-X syndrome has been shown to be closely linked also to a Taq I restriction fragment length polymorphism detected by a cDNA probe for factor IX, and the latter locus has been mapped to the subtelomeric region Xq26-Xq28 [Camerino, G., Mattei, M. G., Mattei, G. F., Jaye, B. & Mandel, J. L. (1983) Nature (London) 306, 701-704]. The in situ hybridization studies reported here provide strong evidence that G6PD is located on the Xq telomeric fragment distal to the fragile site. These observations and the well-established knowledge that the genes for Deutan and Protan colorblindness are closely linked to G6PD, but segregate independently of factor IX deficiency, suggest that the fragile site associated with this type of X-linked mental retardation occurs in a region prone to high frequency of meiotic recombination. Images PMID:6595664

  20. Mosaicism in female hybrid hares heterozygous for glucose-6-phosphate dehydrogenase. V. The recovery of DNA synthesis of hare fibroblasts after ultraviolet irradiation

    SciTech Connect

    Janakidevi, K.; Lee, K.T.; Thomas, W.A.; Kroms, M.; Murray, C.D.

    1984-12-01

    The effect of uv irradiation on the recovery of DNA synthesis is examined in a population of hare fibroblasts exhibiting heterozygosity with reference to the X-linked enzyme, glucose-6-phosphate-dehydrogenase (G-6-PD). These cells have been grown from skin explants of a hybrid female cross between Lepus timidus (female) and L. europaeus (male), the former carrying the G-6-PD gene for the slow-moving T variant and the latter with the fast-moving E variant gene. The hybrid, therefore, exhibits genetic mosaicism due to random inactivation in each cell, of one of the two X chromosomes in the embryonic stage. Exponentially growing cells from 13 fibroblast strains, comprising a wide range of E to T ratios, were exposed to moderately low dose of uv irradiation (6 J/m2). The recovery in DNA synthesis during the 2- to 8-h postirradiation period was calculated as the mean percentage rates of (/sup 3/H)thymidine incorporated during the time as compared to the unirradiated zero-time controls. The results show a statistically significant positive correlation as determined by linear regression analysis between the levels of E and the rate of recovery in DNA synthesis. This is valid also at the higher dose of uv (21 J/m2). These results strengthen our earlier observations with 25-hydroxycholesterol that in the in vitro system the cell expressing the E variant is perhaps more resistant to cytotoxic agents. This also indicates that various factors contribute to the development of monotypism which include cell growth, cell death, mutation, and selection, to name a few.

  1. 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

  2. UDP-glucose pyrophosphorylase influences polysaccharide synthesis, cell wall components, and hyphal branching in Ganoderma lucidum via regulation of the balance between glucose-1-phosphate and UDP-glucose.

    PubMed

    Li, Mengjiao; Chen, Tianxi; Gao, Tan; Miao, Zhigang; Jiang, Ailiang; Shi, Liang; Ren, Ang; Zhao, Mingwen

    2015-09-01

    UDP-glucose pyrophosphorylase (UGP) is a key enzyme involved in carbohydrate metabolism, but there are few studies on the functions of this enzyme in fungi. The ugp gene of Ganoderma lucidum was cloned, and enzyme kinetic parameters of the UGP recombinant protein were determined in vitro, revealing that this protein was functional and catalyzed the reversible conversion between Glc-1-P and UDP-Glc. ugp silencing by RNA interference resulted in changes in the levels of the intermediate metabolites Glc-1-P and UDP-Glc. The compounds and structure of the cell wall in the silenced strains were also altered compared with those in the wild-type strains. Moreover, the number of hyphal branches was also changed in the silenced strains. To verify the role of UGP in hyphal branching, a ugp-overexpressing strain was constructed. The results showed that the number of hyphal branches was influenced by UGP. The mechanism underlying hyphal branching was further investigated by adding exogenous Glc-1-P. Our results showed that hyphal branching was regulated by a change in the cytosolic Ca(2+) concentration, which was affected by the level of the intermediate metabolite Glc-1-P, in G. lucidum. Our findings indicate the existence of an interaction between carbon metabolism and Ca(2+) signaling in this fungus. PMID:26235043

  3. Biochemical characterization of recombinant phosphoglucose isomerase of Mycobacterium tuberculosis

    SciTech Connect

    Mathur, Divya; Ahsan, Zaid; Tiwari, Madhulika; Garg, Lalit C. . E-mail: lalitcgarg@yahoo.com

    2005-11-18

    Phosphoglucose isomerase (PGI) is a well-characterized ubiquitous enzyme involved in the glycolytic pathway. It catalyzes the reversible isomerization of D-glucopyranose-6-phosphate and D-fructofuranose-6-phosphate and is present in all living cells. However, there is interspecies variation at the level of the primary structure which sometimes produces heterogeneity at the structural and functional levels. In order to evaluate and characterize the mycobacterial PGI, the gene encoding the PGI from Mycobacterium tuberculosis H37Rv was cloned in pET-22b(+) vector and expressed in Escherichia coli. The target DNA was PCR amplified from the bacterial artificial chromosome using specific primers and cloned under the control of T7 promoter. Upon induction with IPTG, the recombinant PGI (rPGI) expressed partly as soluble protein and partly as inclusion bodies. The rPGI from the soluble fraction was purified to near homogeneity by ion-exchange chromatography. Mass spectrum analysis of the purified rPGI revealed its mass to be 61.45 kDa. The purified rPGI was enzymatically active and the specific activity was 600 U/mg protein. The K {sub m} of rPGI was determined to be 0.318 mM for fructose-6-phosphate and the K {sub i} was 0.8 mM for 6-phosphogluconate. The rPGI exhibited optimal activity at 37 deg C and pH 9.0, and did not require mono- or divalent cations for its activity.

  4. Affinity labeling and characterization of the active site histidine of glucosephosphate isomerase

    SciTech Connect

    Gibson, D.R.; Gracy, R.W.; Hartman, F.C.

    1980-10-10

    N-bromoacetylethanolamine phosphate was found to act as a specific affinity label for the active center of glucosephosphate isomerase. The inactivation process followed pseudo-first order kinetics, was irreversible, and exhibited rate saturation kinetics with minimal half-lives of inactivation of 4.5 and 6.3 min for the enzyme isolated from human placenta and rabbit muscle, respectively. The pH dependence of the inactivation process closely paralleled the pH dependence of the overall catalytic process with pK/sub a/ values at pH 6.4 and 9.0. The stoichiometry of labeling of either enzyme, as determined with N-bromo(/sup 14/C/sub 2/)acetylethanolamine phosphate, was 1 eq of the affinity label/subunit of enzyme. After acid hydrolysis and amino acid analysis of the radioactive affinity-labeled human enzyme, only radioactive 3-carboxymethyl histidine was found. In the case of the rabbit enzyme, the only radioactive derivative obtained was 1-carboxymethyl histidine. Active site tryptic peptides were isolated by solvent extraction, thin layer peptide fingerprinting, and ion exchange chromatography before and after removal of the phosphate from the active site peptide. Amino acid analysis of the labeled peptides from the two species were very similar. Using high sensitivity methods for sequence analysis, the primary structure of the active site was established as Val-Leu-His-Ala-Glu-Asn-Val-Asp (Gly,Thr,Ser) Glu-Ile (Thr-Gly-His-Lys-Glx)-Tyr-Phe. Apparent sequence homology between the catalytic center of glucosephosphate isomerase and triosephosphate isomerase suggest that the two enzymes may have evolved from a common ancestral gene.

  5. 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.

  6. The effects of triton X-100 on the transfer of mannose, glucose and n-acetylglusomine phosphate to dolichol monophosphate by preparations of rough and smooth endoplasmic reticulum and of mitochondria of rat liver.

    PubMed Central

    Coolbear, T; Mookerjea, S; Hemming, F W

    1979-01-01

    Triton X-100 and exogenous dolichol monophosphate have been used to investigate the nature of enzymes responsible for the transfer of mannose, glucose and N-acetylglucosamine phosphate from nucleotide donors to dolichol monophosphate in vesicles derived from rough and smooth endoplasmic reticulum and mitochondria. Mitochondria were shown to contain the highest specific activities of these enzymes. The responses of the glycosyltransferases to increasing concentrations of Triton X-100 and the effect on these responses of exogenous dolichol monophosphate suggest that the enzymes for mannose and glucose transfer are less hydrophobic, and therefore less intrinsic, in the membrane than the enzyme for N-acetylglucosamine phosphate transfer. In smooth vesicles the results are consistent with mannosyl- and glucosyl-transferases being located at both inner and outer faces of the membrane. In rough vesicles and in mitochondria mannosyl- and glucosyl-transferases were confirmed at the outer face. There is, however, only one site of N-acetylglucosamine phosphate transfer, this being more hydrophobically located in the membrane than the other sites of glycosyl transfer. Mitochondrial enzyme activity closely resembled that of rough endoplasmic reticulum in response to Triton X-100 and exogenous dolichol monophosphate, and is probably associated with the outer membrane. PMID:534537

  7. Metabolic phenotype of phosphoglucose isomerase mutants of Corynebacterium glutamicum.

    PubMed

    Marx, Achim; Hans, Stephan; Möckel, Bettina; Bathe, Brigitte; de Graaf, Albert A; McCormack, Ashling C; Stapleton, Cliona; Burke, Kevin; O'Donohue, Michael; Dunican, L K

    2003-09-01

    A series of experiments reported in the literature using fluxomics as an efficient functional genomics tool revealed that the L-lysine production of the Corynebacterium glutamicum strain MH20-22B correlates with the extent of intracellular NADPH supply. Some alternative metabolic engineering strategies to increase intracellular NADPH supply in the C. glutamicum strain DSM5715 were considered and finally the redirection of carbon flux through the pentose phosphate pathway with two NADPH generating enzymatic reactions was favored. Elsewhere, the construction of a phosphoglucose isomerase (Pgi) null mutant of the C. glutamicum strain DSM5715 has been described by utilizing genetic engineering as well as some aspects of its metabolic phenotype. Most interestingly, it was shown that not only could the L-lysine formation be increased by 1.7-fold but the by-product concentration for the null mutant strain was also able to be drastically reduced. In this publication we discuss this metabolic phenotype in detail and present additional data on by-product formation as well as yield considerations. Results from isotope based metabolic flux analysis in combination with considerations on NADPH metabolism clearly exclude the existence of Pgi isoenzymes in C. glutamicum strain DSM5715. The genome region containing the pgi gene was analyzed. It cannot be excluded that polar effects might have been caused by the disruption of the pgi gene and might have contributed to the observed metabolic phenotype of C. glutamicum Pgi mutants. We illustrate growth characteristics of a Pgi mutant of an industrial L-lysine production strain. A reduced growth rate and a biphasic growth behavior was observed. The importance of NADPH reoxidation for well balanced growth in Pgi mutants is discussed. Another phosphoglucose isomerase mutant of C. glutamicum has been described in literature with which an increase in L-lysine yield from 42 to 52% was observed. This finding highlights the general potential

  8. Glucose-6-phosphate dehydrogenase deficiency

    MedlinePlus

    G6PD deficiency; Hemolytic anemia due to G6PD deficiency; Anemia - hemolytic due to G6PD deficiency ... Gallagher PG. Hemolytic anemias. In: Goldman L, Schafer AI, eds. Goldman's Cecil Medicine . 25th ed. Philadelphia, PA: Elsevier Saunders; 2016:chap 161. Janz ...

  9. Glucose-6-phosphate dehydrogenase deficiency

    MedlinePlus

    G-6-PD deficiency; Hemolytic anemia due to G6PD deficiency; Anemia - hemolytic due to G6PD deficiency ... Churchill Livingston; 2008:chap 45. Golan DER. Hemolytic anemias: red cell membrane and metabolic defects. In: Goldman ...

  10. Plastidic Phosphoglucose Isomerase Is an Important Determinant of Starch Accumulation in Mesophyll Cells, Growth, Photosynthetic Capacity, and Biosynthesis of Plastidic Cytokinins in Arabidopsis

    PubMed Central

    De Diego, Nuria; Muñoz, Francisco J.; Baroja-Fernández, Edurne; Li, Jun; Ricarte-Bermejo, Adriana; Baslam, Marouane; Aranjuelo, Iker; Almagro, Goizeder; Humplík, Jan F.; Novák, Ondřej; Spíchal, Lukáš; Doležal, Karel; Pozueta-Romero, Javier

    2015-01-01

    Phosphoglucose isomerase (PGI) catalyzes the reversible isomerization of glucose-6-phosphate and fructose-6-phosphate. It is involved in glycolysis and in the regeneration of glucose-6-P molecules in the oxidative pentose phosphate pathway (OPPP). In chloroplasts of illuminated mesophyll cells PGI also connects the Calvin-Benson cycle with the starch biosynthetic pathway. In this work we isolated pgi1-3, a mutant totally lacking pPGI activity as a consequence of aberrant intron splicing of the pPGI encoding gene, PGI1. Starch content in pgi1-3 source leaves was ca. 10-15% of that of wild type (WT) leaves, which was similar to that of leaves of pgi1-2, a T-DNA insertion pPGI null mutant. Starch deficiency of pgi1 leaves could be reverted by the introduction of a sex1 null mutation impeding β-amylolytic starch breakdown. Although previous studies showed that starch granules of pgi1-2 leaves are restricted to both bundle sheath cells adjacent to the mesophyll and stomata guard cells, microscopy analyses carried out in this work revealed the presence of starch granules in the chloroplasts of pgi1-2 and pgi1-3 mesophyll cells. RT-PCR analyses showed high expression levels of plastidic and extra-plastidic β-amylase encoding genes in pgi1 leaves, which was accompanied by increased β-amylase activity. Both pgi1-2 and pgi1-3 mutants displayed slow growth and reduced photosynthetic capacity phenotypes even under continuous light conditions. Metabolic analyses revealed that the adenylate energy charge and the NAD(P)H/NAD(P) ratios in pgi1 leaves were lower than those of WT leaves. These analyses also revealed that the content of plastidic 2-C-methyl-D-erythritol 4-phosphate (MEP)-pathway derived cytokinins (CKs) in pgi1 leaves were exceedingly lower than in WT leaves. Noteworthy, exogenous application of CKs largely reverted the low starch content phenotype of pgi1 leaves. The overall data show that pPGI is an important determinant of photosynthesis, energy status, growth

  11. Mixed glucose and lactate uptake by Corynebacterium glutamicum through metabolic engineering.

    PubMed

    Neuner, Andreas; Heinzle, Elmar

    2011-03-01

    The Corynebacterium glutamicum ATCC 13032 lysC(fbr) strain was engineered to grow fast on racemic mixtures of lactate and to secrete lysine during growth on lactate as well as on mixtures of lactate and glucose. The wild-type C. glutamicum only grows well on L-lactate. Overexpression of D-lactate dehydrogenase (dld) achieved by exchanging the native promoter of the dld gene for the stronger promoter of the sod gene encoding superoxide dismutase in C. glutamicum resulted in a duplication of biomass yield and faster growth without any secretion of lysine. Elementary mode analysis was applied to identify potential targets for lysine production from lactate as well as from mixtures of lactate and glucose. Two targets for overexpression were pyruvate carboxylase and malic enzyme. The overexpression of these genes using again the sod promoter resulted in growth-associated production of lysine with lactate as sole carbon source with a carbon yield of 9% and a yield of 15% during growth on a lactate-glucose mixture. Both substrates were taken up simultaneously with a slight preference for lactate. As surmised from the elementary mode analysis, deletion of glucose-6-phosphate isomerase resulted in a decreased production of lysine on the mixed substrate. Elementary mode analysis together with suitable objective functions has been found a very useful tool guiding the design of strains producing lysine on mixed substrates. PMID:21370474

  12. High prevalence of hemoglobin disorders and glucose-6-phosphate dehydrogenase (G6PD) deficiency in the Republic of Guinea (West Africa).

    PubMed

    Millimono, Tamba S; Loua, Kovana M; Rath, Silvia L; Relvas, Luis; Bento, Celeste; Diakite, Mandiou; Jarvis, Martin; Daries, Nathalie; Ribeiro, Leticia M; Manco, Licínio; Kaeda, Jaspal S

    2012-01-01

    Reliable and accurate epidemiological data is a prerequisite for a cost effective screening program for inherited disorders, which however, is lacking in a number of developing countries. Here we report the first detailed population study in the Republic of Guinea, a sub-Saharan West African country, designed to assess the frequency of glucose-6-phosphate dehydrogenase (G6PD) deficiency and hemoglobinopathies, including screening for thalassemia. Peripheral blood samples from 187 Guinean adults were screened for hemoglobin (Hb) variants by standard hematological methods. One hundred and ten samples from males were screened for G6PD deficiency by the fluorescent spot test. Molecular analysis was performed for the most common α-thalassemia (α-thal) deletions, β-globin gene mutations, G6PD variants B (376A), A (376G), A- (376G/202A) and Betica (376G/968C), using polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP) or sequencing. Of the 187 subjects screened, 36 were heterozygous for Hb S [β6(A3)Glu→Val, GAG>GTG] (allele frequency 9.62%). Sixty-four subjects were heterozygous and seven were homozygous for the -α(3.7) kb deletion (allele frequency 20.85%). β-Thalassemia alleles were detected in five subjects, four with the -29 (A>G) mutation (allele frequency 1.07%) and one with codon 15 (TGG>TAG) (allele frequency 0.96%). The G6PD A- and G6PD Betica deficient variants were highly prevalent with a frequency of 5.7 and 3.3%, respectively. While we did not test for ferritin levels or α(0)-thal, four females (5.2%) had red cell indices strongly suggestive of iron deficient anemia: Hb <9.7 g/dL; MCH <19.3 pg; MCV <68.2; MCHC <31.6 g/dl; RDW >19.8%. Our results are consistent with high frequency of alleles such as Hb S, α-thal and G6PD deficient alleles associated with malaria resistance. Finding a 9.6% Hb S allele frequency supports the notion for a proficient neonatal screening to identify the sickle cell patients, who might benefit

  13. Biosynthesis of Dolichyl Phosphate

    PubMed Central

    Hopp, H. Esteban; Daleo, Gustavo R.; Romero, Pedro A.; Lezica, Rafael Pont

    1978-01-01

    This is the first report not only on the presence of polyprenyl phosphates and their site of synthesis in algae, but also on the formation of their sugar derivatives in this system. A glucose acceptor lipid was isolated from the nonphotosynthetic alga Prototheca zopfii. The lipid was acidic and resistant to mild acid and alkaline treatments. The glucosylated lipid was labile to mild acid hydrolysis and resistant to phenol treatment and catalytic hydrogenation, as dolichyl phosphate glucose is. These results are consistent with the properties of an α-saturated polyprenyl phosphate. The polyprenylic nature of the lipid was confirmed by biosynthesis from radioactive mevalonate. The [14C]lipid had the same chromatographic properties as dolichyl phosphate in DEAE-cellulose and Sephadex LH-20. Strong alkaline treatment and enzymic hydrolysis liberated free alcohols with chain lengths ranging from C90 to C105, C95 and C100 being the most abundant molecular forms. The glucose acceptor activity of the biosynthesized polyprenyl phosphate was confirmed. The ability of different subcellular fractions to synthesize dolichyl phosphate was studied. Mitochondria and the Golgi apparatus were the sites of dolichyl phosphate synthesis from mevalonate. PMID:16660269

  14. Human triosephosphate isomerase deficiency resulting from mutation of Phe-240

    SciTech Connect

    Minling Chang; Xiaoyun Wu; Maquat, L.E. ); Artymiuk, P.J. ); Hollan, S. ); Lammi, A. )

    1993-06-01

    Triosephosphate isomerase (TPI; D-glyceraldehyde-3-phosphate ketolisomerase [E.C.5.3.1.1]) deficiency is an autosomal recessive disorder that typically results in chronic, nonspherocytic hemolytic anemia and in neuromuscular impairment. The molecular basis of this disease was analyzed for one Hungarian family and for two Australian families by localizing the defects in TPI cDNA and by determining how each defect affects TPI gene expression. The Hungarian family is noteworthy in having the first reported case of an individual, A. Jo., who harbors two defective TPI alleles but who does not manifest neuromuscular disabilities. This family was characterized by two mutations that have never been described. One is a missense mutation within codon 240 (TTC [Phe][r arrow]CTC [Leu]), which creates a thermolabile protein, as indicated by the results of enzyme activity assays using cell extracts. This substitution, which changes a phylogenetically conserved amino acid, may affect enzyme activity by dusrupting intersubunit contacts or substrate binding, as deduced from enzyme structural studies. The other mutation has yet to be localized but reduces the abundance of TPI mRNA 10--20-fold. Each of the Australian families was characterized by a previously described mutation within codon 104 (GAG [Glu][r arrow]GAC [Asp]), which also results in thermolabile protein. 49 refs., 6 figs., 1 tab.

  15. Psychrophilic, mesophilic, and thermophilic triosephosphate isomerases from three clostridial species.

    PubMed Central

    Shing, Y W; Akagi, J M; Himes, R H

    1975-01-01

    Triosephosphate isomerase was purified to homogeneity as judged by analytical gel electrophoresis from clostridium sp. strain 69, clostridium pasteurianum, and C. thermosaccharolyticum, which grow optimally at 18, 37, and 55 C, respectively. Comparative studies on these purified proteins showed that they had the same molecular weight (53,000) and subunit molecular weight (26,500). They were equally susceptible to the active site-directed inhibitor, glycidol phosphate. However, their temperature and pH optima, as well as their stabilities to heat, urea, and sodium dodecyl sulfate, differed. The proteins also had different mobilities in acrylamide gel electrophoresis. This difference in ionic character was also reflected in the elution behavior of the enzymes from hydroxyapatite and in the isoelectric points determined by isoelectric focusing in acrylamide gel. The amino acid composition of these proteins showed that the thermophilic enzyme contains a greater amount of proline than the other enzymes. The ratio of acidic amino acids to basic amino acids was 1.79, 1.38, and 1.66 for the thermophilic mesophilic and psychrophilic enzymes, respectively. This is consistent with the relative isoelectric point values of these three enzymes. Images PMID:235509

  16. Physiological and Pathogenic Roles of Prolyl Isomerase Pin1 in Metabolic Regulations via Multiple Signal Transduction Pathway Modulations.

    PubMed

    Nakatsu, Yusuke; Matsunaga, Yasuka; Yamamotoya, Takeshi; Ueda, Koji; Inoue, Yuki; Mori, Keiichi; Sakoda, Hideyuki; Fujishiro, Midori; Ono, Hiraku; Kushiyama, Akifumi; Asano, Tomoichiro

    2016-01-01

    Prolyl isomerases are divided into three groups, the FKBP family, Cyclophilin and the Parvulin family (Pin1 and Par14). Among these isomerases, Pin1 is a unique prolyl isomerase binding to the motif including pSer/pThr-Pro that is phosphorylated by kinases. Once bound, Pin1 modulates the enzymatic activity, protein stability or subcellular localization of target proteins by changing the cis- and trans-formations of proline. Several studies have examined the roles of Pin1 in the pathogenesis of cancers and Alzheimer's disease. On the other hand, recent studies have newly demonstrated Pin1 to be involved in regulating glucose and lipid metabolism. Interestingly, while Pin1 expression is markedly increased by high-fat diet feeding, Pin1 KO mice are resistant to diet-induced obesity, non-alcoholic steatohepatitis and diabetic vascular dysfunction. These phenomena result from the binding of Pin1 to several key factors regulating metabolic functions, which include insulin receptor substrate-1, AMPK, Crtc2 and NF-κB p65. In this review, we focus on recent advances in elucidating the physiological roles of Pin1 as well as the pathogenesis of disorders involving this isomerase, from the viewpoint of the relationships between signal transductions and metabolic functions. PMID:27618008

  17. Agrobacterium and biolistic transformation of onion using non-antibiotic selection marker phosphomannose isomerase.

    PubMed

    Aswath, Chenna Reddy; Mo, Sung Youn; Kim, Doo Hwan; Park, S Won

    2006-03-01

    A new selection system for onion transformation that does not require the use of antibiotics or herbicides was developed. The selection system used the Escherichia coli gene that encodes phosphomannose isomerase (pmi). Transgenic plants carrying the manA gene that codes for pmi can detoxify mannose-6-phosphate by conversion to fructose-6-phosphate, an intermediate of glycolysis, via the pmi activity. Six-week-old embryogenic callus initiated from seedling radicle was used for transformation. Transgenic plants were produced efficiently with transformation rates of 27 and 23% using Agrobacterium and biolistic system, respectively. Untransformed shoots were eliminated by a stepwise increase from 10 g l(-1) sucrose with 10 g l(-1) mannose in the first selection to only 10 g l(-1) mannose in the second selection. Integrative transformation was confirmed by PCR, RT-PCR and Southern hybridization. PMID:16211408

  18. Arabidopsis Phosphomannose Isomerase 1, but Not Phosphomannose Isomerase 2, Is Essential for Ascorbic Acid Biosynthesis*S⃞

    PubMed Central

    Maruta, Takanori; Yonemitsu, Miki; Yabuta, Yukinori; Tamoi, Masahiro; Ishikawa, Takahiro; Shigeoka, Shigeru

    2008-01-01

    We studied molecular and functional properties of Arabidopsis phosphomannose isomerase isoenzymes (PMI1 and PMI2) that catalyze reversible isomerization between d-fructose 6-phosphate and d-mannose 6-phosphate (Man-6P). The apparent Km and Vmax values for Man-6P of purified recombinant PMI1 were 41.3 ± 4.2 μm and 1.89 μmol/min/mg protein, respectively, whereas those of purified recombinant PMI2 were 372 ± 13 μm and 22.5 μmol/min/mg protein, respectively. Both PMI1 and PMI2 were inhibited by incubation with EDTA, Zn2+, Cd2+, and l-ascorbic acid (AsA). Arabidopsis PMI1 protein was constitutively expressed in both vegetative and reproductive organs under normal growth conditions, whereas the PMI2 protein was not expressed in any organs under light. The induction of PMI1 expression and an increase in the AsA level were observed in leaves under continuous light, whereas the induction of PMI2 expression and a decrease in the AsA level were observed under long term darkness. PMI1 showed a diurnal expression pattern in parallel with the total PMI activity and the total AsA content in leaves. Moreover, a reduction of PMI1 expression through RNA interference resulted in a substantial decrease in the total AsA content of leaves of knockdown PMI1 plants, whereas the complete inhibition of PMI2 expression did not affect the total AsA levels in leaves of knock-out PMI2 plants. Consequently, this study improves our understanding of the molecular and functional properties of Arabidopsis PMI isoenzymes and provides genetic evidence of the involvement of PMI1, but not PMI2, in the biosynthesis of AsA in Arabidopsis plants. PMID:18755683

  19. 13C and 31P chemical shielding tensors of a single crystal of dipotassium α- D-glucose-1-phosphate dihydrate. An application of a 13C-{ 1H, 31P} triple-resonance technique

    NASA Astrophysics Data System (ADS)

    McDowell, C. A.; Naito, A.; Sastry, D. L.; Takegoshi, K.

    The 13C NMR spectra of a single crystal of dipotassium α- D-glucose-l-phosphate dehydrate for different orientations in the external magnetic field, were recorded by using 1H and 31P double nuclear decoupling. To overcome difficulties encountered because of the high 13C RF power required to achieve the Hartmann-Hahn condition, a new cross-polarization method (K. Takegoshi and C. A. McDowell, J. Magn. Reson.67, 356 (1986)) was used. The directions of the most shielded principal value of the 13C chemical shielding tensors for the C2-C6 carbon nuclei in the glucose group were along the CO bond, and that for the CI carbon nucleus made an angle of 42† with the C1-O5 bond direction in the O1-C1-O5 plane. The 31P chemical shielding tensors are axially symmetric and the direction of the least shielded principal value is almost parallel to the P-O1(R) bond, which is the longest among the four PO bonds in the phosphate moiety.

  20. 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

  1. Phosphomannose Isomerase Inhibitors Improve N-Glycosylation in Selected Phosphomannomutase-deficient Fibroblasts*

    PubMed Central

    Sharma, Vandana; Ichikawa, Mie; He, Ping; Bravo, Yalda; Dahl, Russell; Ng, Bobby G.; Cosford, Nicholas D. P.; Freeze, Hudson H.

    2011-01-01

    Congenital disorders of glycosylation (CDG) are rare genetic disorders due to impaired glycosylation. The patients with subtypes CDG-Ia and CDG-Ib have mutations in the genes encoding phosphomannomutase 2 (PMM2) and phosphomannose isomerase (MPI or PMI), respectively. PMM2 (mannose 6-phosphate → mannose 1-phosphate) and MPI (mannose 6-phosphate ⇔ fructose 6-phosphate) deficiencies reduce the metabolic flux of mannose 6-phosphate (Man-6-P) into glycosylation, resulting in unoccupied N-glycosylation sites. Both PMM2 and MPI compete for the same substrate, Man-6-P. Daily mannose doses reverse most of the symptoms of MPI-deficient CDG-Ib patients. However, CDG-Ia patients do not benefit from mannose supplementation because >95% Man-6-P is catabolized by MPI. We hypothesized that inhibiting MPI enzymatic activity would provide more Man-6-P for glycosylation and possibly benefit CDG-Ia patients with residual PMM2 activity. Here we show that MLS0315771, a potent MPI inhibitor from the benzoisothiazolone series, diverts Man-6-P toward glycosylation in various cell lines including fibroblasts from CDG-Ia patients and improves N-glycosylation. Finally, we show that MLS0315771 increases mannose metabolic flux toward glycosylation in zebrafish embryos. PMID:21949237

  2. 13C NMR study of the generation of C2- and C3-deuterated lactic acid by tumoral pancreatic islet cells exposed to D-[1-13C]-, D-[2-13C]- and D-[6-13C]-glucose in 2H2O.

    PubMed

    Willem, R; Biesemans, M; Kayser, F; Malaisse, W J

    1994-03-01

    Tumoral pancreatic islet cells of the RIN5mF line were incubated for 120 min in media prepared in 2H2O and containing D-[1-13C]glucose, D-[2-13C]glucose, and D-[6-13C]glucose. The generation of C2- and C3-deuterated lactic acid was assessed by 13C NMR. The interpretation of experimental results suggests that a) the efficiency of deuteration on the C1 of D-fructose 6-phosphate does not exceed about 47% and 4% in the phosphoglucoisomerase and phosphomannoisomerase reactions, respectively; b) approximately 38% of the molecules of D-glyceraldehyde 3-phosphate generated from D-glucose escape deuteration in the sequence of reactions catalyzed by triose phosphate isomerase and aldolase; and c) about 41% of the molecules of pyruvate generated by glycolysis are immediately converted to lactate, the remaining 59% of pyruvate molecules undergoing first a single or double back-and-forth interconversion with L-alanine. It is proposed that this methodological approach, based on high resolution 13C NMR spectroscopy, may provide novel information on the regulation of back-and-forth interconversion of glycolytic intermediates in intact cells as modulated, for instance, by enzyme-to-enzyme tunneling. PMID:8057796

  3. Isotopic exchange plus substrate and inhibition kinetics of D-xylose isomerase do not support a proton-transfer mechanism.

    PubMed

    Allen, K N; Lavie, A; Farber, G K; Glasfeld, A; Petsko, G A; Ringe, D

    1994-02-15

    The D-xylose isomerase of Streptomyces olivochromogenes is a Mg2+- or Mn(2+)-dependent enzyme that catalyzes the aldose-ketose isomerization of xylose to xylulose or of glucose to fructose. Proton exchange into water during enzyme-catalyzed isomerization of C-2 tritiated glucose at 15, 25 and 55 degrees C shows < 0.6% exchange (the loss of one proton in every billion turnovers). High concentrations of guanidine hydrochloride and extremes of pH had no effect on the amount of exchange detected. Such a low percentage of exchange is inconsistent with a proton-transfer mechanism as the main kinetic pathway for isomerization. 19F NMR experiments showed no release of fluoride after incubation of the enzyme for 4 weeks with 800 mM 3-deoxy-3-fluoroglucose or 3-deoxy-3-fluoroallose (both are competitive inhibitors with Ki values of 600 mM). This result is also inconsistent with a proton-transfer mechanism. A hydride-shift mechanism following ring opening has been proposed for the isomerization. Enzyme-catalyzed ring opening was directly measured by demonstrating H2S release upon reaction of xylose isomerase with 1-thioglucose. D-Xylose isomerase-catalyzed interconversion of glucose to fructose exhibited linear Arrhenius behavior with an activation energy of 14 kcal/mol from 0 to 50 degrees C. No change in rate-determining step occurs over this temperature range. 13C NMR experiments with glucose show that enzyme-bound magnesium or manganese does not interact specifically with any one site on the sugar. These results are consistent with nonproductive binding modes for the substrate glucose in addition to productive binding. PMID:8312268

  4. Ethanol production from lignocellulosic hydrolysates using engineered Saccharomyces cerevisiae harboring xylose isomerase-based pathway.

    PubMed

    Ko, Ja Kyong; Um, Youngsoon; Woo, Han Min; Kim, Kyoung Heon; Lee, Sun-Mi

    2016-06-01

    The efficient co-fermentation of glucose and xylose is necessary for the economically feasible bioethanol production from lignocellulosic biomass. Even with xylose utilizing Saccharomyces cerevisiae, the efficiency of the lignocellulosic ethanol production remains suboptimal mainly due to the low conversion yield of xylose to ethanol. In this study, we evaluated the co-fermentation performances of SXA-R2P-E, a recently engineered isomerase-based xylose utilizing strain, in mixed sugars and in lignocellulosic hydrolysates. In a high-sugar fermentation with 70g/L of glucose and 40g/L of xylose, SXA-R2P-E produced 50g/L of ethanol with an yield of 0.43gethanol/gsugars at 72h. From dilute acid-pretreated hydrolysates of rice straw and hardwood (oak), the strain produced 18-21g/L of ethanol with among the highest yield of 0.43-0.46gethanol/gsugars ever reported. This study shows a highly promising potential of a xylose isomerase-expressing strain as an industrially relevant ethanol producer from lignocellulosic hydrolysates. PMID:26990396

  5. Characterization of triosephosphate isomerase from Mycoplasma gallisepticum.

    PubMed

    Bao, Shijun; Chen, Danqing; Yu, Shengqing; Chen, Hongjun; Tan, Lei; Hu, Meirong; Qiu, Xusheng; Song, Cuiping; Ding, Chan

    2015-09-01

    Triosephosphate isomerase (Tpi) is a glycolytic enzyme that is essential for efficient energy production in many pathogens. However, its function in Mycoplasma gallisepticum has not been fully elucidated. In this study, the mga0357 gene of M. gallisepticum, which encodes TpiA (MGTpiA), was amplified and expressed in Escherichia coli by IPTG induction. The purified recombinant MGTpiA protein exhibited catalytic activity that was similar to TPI from rabbit muscle, reducing NAD(+) to NADH. The MGTpiA was also found to be a surface-exposed protein by western blotting and immunofluorescence assays. In addition, cytadherence inhibition assays confirmed that the cytadherence of M. gallisepticum to the DF-1 cells was significantly inhibited by the anti-MGTpiA serum. The results of the study suggested that MGTpiA plays an important role in the metabolism and closely related to the M. gallisepticum pathogenicity. PMID:26319024

  6. 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. PMID:26820767

  7. Targeted disruption of the housekeeping gene encoding glucose 6-phosphate dehydrogenase (G6PD): G6PD is dispensable for pentose synthesis but essential for defense against oxidative stress.

    PubMed Central

    Pandolfi, P P; Sonati, F; Rivi, R; Mason, P; Grosveld, F; Luzzatto, L

    1995-01-01

    Glucose 6-phosphate dehydrogenase (G6PD) is a housekeeping enzyme encoded in mammals by an X-linked gene. It has important functions in intermediary metabolism because it catalyzes the first step in the pentose phosphate pathway and provides reductive potential in the form of NADPH. In human populations, many mutant G6PD alleles (some present at polymorphic frequencies) cause a partial loss of G6PD activity and a variety of hemolytic anemias, which vary from mild to severe. All these mutants have some residual enzyme activity, and no large deletions in the G6PD gene have ever been found. To test which, if any, function of G6PD is essential, we have disrupted the G6PD gene in male mouse embryonic stem cells by targeted homologous recombination. We have isolated numerous clones, shown to be recombinant by Southern blot analysis, in which G6PD activity is undetectable. We have extensively characterized individual clones and found that they are extremely sensitive to H2O2 and to the sulfydryl group oxidizing agent, diamide. Their markedly impaired cloning efficiency is restored by reducing the oxygen tension. We conclude that G6PD activity is dispensable for pentose synthesis, but is essential to protect cells against even mild oxidative stress. Images PMID:7489710

  8. Enzymatic, spectrophotometric determination of glucose, fructose, sucrose, and inulin/oligofructose in foods.

    PubMed

    Steegmans, Monique; Iliaens, Saskia; Hoebregs, Hubert

    2004-01-01

    A fast, simple, and accurate method, using only standard laboratory equipment, was developed for the quantification of glucose, fructose, sucrose, and inulin/oligofructose in different food matrixes. Samples were extracted using boiling water and hydrolyzed with sucrase and fructanase. Sugars were determined in the initial extract and in both hydrolysates using an enzymatic, spectrophotometric kit for glucose and fructose determination with hexokinase, glucose-6-phosphate dehydrogenase, and phosphoglucose isomerase. Calculations of sucrose and inulin/oligofructose were based only on fructose measurement. Glucose results of the hydrolysates were not used for inulin/oligofructose calculations because of possible interference. Released glucose by the hydrolysis of maltose or by possible partial hydrolysis of other compounds like maltodextrines, starch, lactose, or maltitol could interfere in the measurement of the sucrase and the fructanase hydrolysates. To validate the method, a wide range of different food matrixes and different amounts of inulin/oligofructose (1-54%) were analyzed. Mean recovery +/- relative standard deviation (RSD) for inulin or oligofructose was 96.0 +/- 5.3%. The RSDr for inulin/oligofructose measured on 35 food samples, analyzed in duplicate, was 5.9%. Accuracy and precision of the method were less for samples with large concentrations of sucrose, maltose, maltodextrines, or starch (ratio to inulin/oligofructose >4 to 1). Precision and accuracy were comparable with those of the ion exchange chromatographic method AOAC 997.08 and the enzymatic, spectrophotometric method AOAC 999.03. In contrast to 999.03, this method allows the accurate quantification of both GFn and Fn forms. PMID:15493679

  9. Correlation between the Suppression of Glucose and Phosphate Uptake and the Release of Protein from Viable Carrot Root Cells Treated with Monovalent Cations

    PubMed Central

    Nieman, R. H.; Willis, Catherine

    1971-01-01

    Treating carrot (Daucus carota L.) discs with ice-cold NaCl solutions for 30 minutes caused three effects that appear to be functionally related: the exchange of tissue Ca2+ and Mg2+ for Na+, the release of protein, and the suppression of active uptake of glucose and orthophosphate. Cyclosis continued apparently unabated after treatment with NaCl at concentrations of up to 0.25 m, so the cells remained viable and energetically competent. The correlation between the release of Ca2+ and Mg2+ and release of protein, and between these effects and the suppression of glucose and orthophosphate uptake, supports the hypothesis that divalent cations maintain, and monovalent cations disrupt, linkages between the outer cell surface and proteins required for active solute uptake. Calcium preserved uptake activity only when it was added in time to prevent the release of protein. Cells gradually recovered some glucose uptake activity after it had been completely inactivated by treatment with 0.25 m NaCl. This recovery occurred in the absence of added Ca2+. It was inhibited by puromycin and so appears to require some protein synthesis. Beet (Beta vulgaris L.) discs were more resistant than carrot discs to treatment with NaCl solutions, thus reflecting the difference in tolerance of the two species to sodicity. PMID:16657783

  10. The in vivo effects of 3-deoxy-3-fluoro-D-glucose metabolism on respiration in Locusta migratoria.

    PubMed

    Romaschin, A; Taylor, N F

    1981-04-01

    The basis of the toxicity of 3-deoxy-fluoro-D-glucose (3FG) in adult Locusta migratoria is examined in vivo by a radiorespirometric analysis of 14CO2 from the locust after injections of 3FG prior to injections of D-[1-14C]glucose, D-[6-14C]glucose, or [1-14C]acetate. The results indicate that 3FG metabolism irreversibly inhibits glycolysis and not the hexose monophosphate pathway or the tricarboxylic acid cycle. It is also established that during metabolism of 3FG fluoride ion is released. Evidence for the metabolism of 3FG in the whole insect as far as triosephosphate isomerase is based on 3H2O release after injections of D-[3-3H]3FG. Further support for the metabolism of 3FG to fluorinated sugar phosphates is provided by chromatographic and 19F MNR analysis of 3FG poisoned locust tissue extracts. Based on these results a biochemical mode of toxicity of 3FG in locusts is discussed. PMID:7018654

  11. Metabolic engineering of Escherichia coli for (2S)-pinocembrin production from glucose by a modular metabolic strategy.

    PubMed

    Wu, Junjun; Du, Guocheng; Zhou, Jingwen; Chen, Jian

    2013-03-01

    Flavonoids are valuable natural products widely used in human health and nutrition. Recent advances in synthetic biology and metabolic engineering have yielded improved strain titers and yields. However, current fermentation strategies often require supplementation of expensive phenylpropanoic precursors in the media and separate evaluation of each strategy in turn as part of the flavonoid pathway, implicitly assuming the modifications are additive. In this study, an Escherichia coli fermentation system was developed to bypass both of these problems. An eight-step pathway, consisting of 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase (DAHPS), chorismate mutase/prephenate dehydratase (CM/PDT), phenylalanine ammonia lyase (PAL), 4-coumarate:CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), malonate synthetase, and malonate carrier protein, was assembled on four vectors in order to produce the flavonoid precursor (2S)-pinocembrin directly from glucose. Furthermore, a modular metabolic strategy was employed to identify conditions that optimally balance the four pathway modules. Once this metabolic balance was achieved, such strains were capable of producing 40.02mg/L (2S)-pinocembrin directly from glucose. These results were attained by culturing engineered cells in minimal medium without additional precursor supplementation. The fermentation platform described here paves the way for the development of an economical process for microbial production of flavonoids directly from glucose. PMID:23246524

  12. Overexpression, purification, crystallization and preliminary diffraction studies of the Protaminobacter rubrum sucrose isomerase SmuA

    SciTech Connect

    Ravaud, Stéphanie; Watzlawick, Hildegard; Haser, Richard; Mattes, Ralf; Aghajari, Nushin

    2006-01-01

    The P. rubrum sucrose isomerase SmuA, a key enzyme in the industrial production of isomaltulose, was crystallized and diffraction data were collected to 1.95 Å resolution. Palatinose (isomaltulose, α-d-glucosylpyranosyl-1,6-d-fructofuranose), a nutritional and acariogenic reducing sugar, is industrially obtained from sucrose by using immobilized cells of Protaminobacter rubrum that produce the sucrose isomerase SmuA. The isomerization of sucrose catalyzed by this enzyme also results in the formation of trehalulose (α-d-glucosylpyranosyl-1,1-d-fructofuranose) in smaller amounts and glucose, fructose and eventually isomaltose as by-products, which lower the yield of the reaction and complicate the recovery of palatinose. The determination of the three-dimensional structure of SmuA will provide a basis for rational protein-engineering studies in order to optimize the industrial production of palatinose. A recombinant form of the 67.3 kDa SmuA enzyme has been crystallized in the native state by the vapour-diffusion method. Crystals belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 61.6, b = 81.4, c = 135.6 Å, and diffract to 1.95 Å resolution on a synchrotron-radiation source.

  13. Phosphate salts

    MedlinePlus

    ... taken by mouth or used as enemas. Indigestion. Aluminum phosphate and calcium phosphate are FDA-permitted ingredients ... Phosphate salts containing sodium, potassium, aluminum, or calcium are LIKELY SAFE for most people when taken by mouth short-term, when sodium phosphate is inserted into the ...

  14. Method for converting sucrose to .beta.-D-glucose

    DOEpatents

    Simmons,; Blake A.; Volponi, Joanne V.; Ingersoll, David; Walker, Andrew

    2009-07-07

    Disclosed is an apparatus and method for continuously converting sucrose to .beta.-D-glucose. The method comprises a three-stage enzymatic reactor in which an aqueous solution of sucrose is first converted into a solution of fructose and .alpha.-D-glucose by passing it through a porous, packed column containing an inert media on which invertase is immobilized. This solution is then sent through a second packed column containing glucose isomerase and finally a third packed column containing mutarotase. Solution temperature and pH are adjusted to maximize glucose output.

  15. The glucose-6-phosphatase system.

    PubMed Central

    van Schaftingen, Emile; Gerin, Isabelle

    2002-01-01

    Glucose-6-phosphatase (G6Pase), an enzyme found mainly in the liver and the kidneys, plays the important role of providing glucose during starvation. Unlike most phosphatases acting on water-soluble compounds, it is a membrane-bound enzyme, being associated with the endoplasmic reticulum. In 1975, W. Arion and co-workers proposed a model according to which G6Pase was thought to be a rather unspecific phosphatase, with its catalytic site oriented towards the lumen of the endoplasmic reticulum [Arion, Wallin, Lange and Ballas (1975) Mol. Cell. Biochem. 6, 75--83]. Substrate would be provided to this enzyme by a translocase that is specific for glucose 6-phosphate, thereby accounting for the specificity of the phosphatase for glucose 6-phosphate in intact microsomes. Distinct transporters would allow inorganic phosphate and glucose to leave the vesicles. At variance with this substrate-transport model, other models propose that conformational changes play an important role in the properties of G6Pase. The last 10 years have witnessed important progress in our knowledge of the glucose 6-phosphate hydrolysis system. The genes encoding G6Pase and the glucose 6-phosphate translocase have been cloned and shown to be mutated in glycogen storage disease type Ia and type Ib respectively. The gene encoding a G6Pase-related protein, expressed specifically in pancreatic islets, has also been cloned. Specific potent inhibitors of G6Pase and of the glucose 6-phosphate translocase have been synthesized or isolated from micro-organisms. These as well as other findings support the model initially proposed by Arion. Much progress has also been made with regard to the regulation of the expression of G6Pase by insulin, glucocorticoids, cAMP and glucose. PMID:11879177

  16. Structural and biochemical characterization of a recombinant triosephosphate isomerase from Rhipicephalus (Boophilus) microplus

    SciTech Connect

    Moraes, Jorge; Arreola, Rodrigo; Cabrera, Nallely; Saramago, Luiz; Freitas, Daniela; Masuda, Aoi; da Silva Vaz Jr., Itabajara; Tuena de Gomez-Puyou, Marietta; Perez-Montfort, Ruy; Gomez-Puyou, Armando; Logullo, Carlos

    2012-02-06

    Triosephosphate isomerase (TIM) is an enzyme with a role in glycolysis and gluconeogenesis by catalyzing the interconversion between glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. This enzyme has been used as a target in endoparasite drug development. In this work we cloned, expressed, purified and studied kinetic and structural characteristics of TIM from tick embryos, Rhipicephalus (Boophilus) microplus (BmTIM). The Km and Vmax of the recombinant BmTIM with glyceraldehyde 3-phosphate as substrate, were 0.47 mM and 6031 {micro}mol min{sup -1} mg protein{sup -1}, respectively. The resolution of the diffracted crystal was estimated to be 2.4 {angstrom} and the overall data showed that BmTIM is similar to other reported dimeric TIMs. However, we found that, in comparison to other TIMs, BmTIM has the highest content of cysteine residues (nine cysteine residues per monomer). Only two cysteines could make disulfide bonds in monomers of BmTIM. Furthermore, BmTIM was highly sensitive to the action of the thiol reagents dithionitrobenzoic acid and methyl methane thiosulfonate, suggesting that there are five cysteines exposed in each dimer and that these residues could be employed in the development of species-specific inhibitors.

  17. Cellular and biochemical characterization of two closely related triosephosphate isomerases from Trichomonas vaginalis.

    PubMed

    Figueroa-Angulo, Elisa E; Estrella-Hernández, Priscila; Salgado-Lugo, Holjes; Ochoa-Leyva, Adrián; Gómez Puyou, Armando; Campos, Silvia S; Montero-Moran, Gabriela; Ortega-López, Jaime; Saab-Rincón, Gloria; Arroyo, Rossana; Benítez-Cardoza, Claudia G; Brieba, Luis G

    2012-11-01

    The glycolytic enzyme triosephosphate isomerase catalyses the isomerization between glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. Here we report that Trichomonas vaginalis contains 2 fully functional tpi genes. Both genes are located in separated chromosomal context with different promoter regulatory elements and encode ORFs of 254 amino acids; the only differences between them are the character of 4 amino acids located in α-helices 1, 2 and 8. Semi-quantitative RT-PCR assays showed that tpi2 transcript is approximately 3·3-fold more abundant than tpi1. Using an anti-TvTIM2 polyclonal antibody it was demonstrated that TIM proteins have a cytoplasmic localization and both enzymes are able to complement an Escherichia coli strain carrying a deletion of its endogenous tpi gene. Both TIM proteins assemble as dimers and their secondary structure assessment is essentially identical to TIM from Saccharomyces cerevisiae. The kinetic catalytic constants of the recombinant enzymes using glyceraldehyde-3-phosphate as substrate are similar to the catalytic constants of TIMs from other organisms including parasitic protozoa. As T. vaginalis depends on glycolysis for ATP production, we speculate 2 possible reasons to maintain a duplicated tpi copy on its genome: an increase in gene dosage or an early event of neofunctionalization of TIM as a moonlighting protein. PMID:22931930

  18. Overexpression of protein disulfide isomerase in Aspergillus.

    PubMed

    El-Adawi, H; Khanh, N Q; Gassen, H

    2000-10-01

    One of the major problems with the production of biotechnologically valuable proteins has been the purification of the product. For Escherichia coli and Saccharomyces cerevisiae, there are several techniques for the purification of intracellular proteins, but these are time consuming and often result in poor yields. Purification can be considerably facilitated, if the product is secreted from the host cell. In the work presented, we have constructed an expression vector (pSGNH2) for the secretion of protein disulfide isomerase (PDI; EC 5.3.4.1) from Aspergillus niger, in which the retention signal His-Asp-Glu-Leu (H-D-E-L) was modified to Ala-Leu-Glu-Gln (A-L-E-Q) via the polymerase chain reaction (PCR) method. The PDI gene was placed under the control of the A. oryzae alpha-amylase promoter. This expression vector was transformed into A. niger NRRL3, resulting in PDI secretion into the medium. The catalytic activity of overexpressed PDI from A. niger was indistinguishable from that of PDI isolated from bovine liver. With further strain improvement and optimization of culture conditions, it could be possible to raise the PDI production to the bioprocessing scale. PMID:10977899

  19. Anomeric specificity of D-xylose isomerase.

    PubMed

    Collyer, C A; Goldberg, J D; Viehmann, H; Blow, D M; Ramsden, N G; Fleet, G W; Montgomery, F J; Grice, P

    1992-12-01

    Crystal structures of complexes of D-xylose isomerase with deoxysugars have been determined. Deoxynojirimycin is a structural analogue of alpha-pyranose and mimics the binding of these aldose substrates. The structure of this complex supports the hypothesis that an imidazole group catalyzes ring opening of the pyranose. The steric restrictions in the active site of the enzyme prevent a beta-pyranose from binding in the same way. For the reverse reaction with ketoses, the anomeric specificity is less certain. Dideoxyimino-D-glucitol is a structural analogue of the ketose alpha-D-furanose. The binding of the inhibitor dideoxyimino-D-glucitol to the crystals of the enzyme does not mimic the binding of the reactive alpha-D-fructofuranose. Superposition of the nonphysiological substrate alpha-D-fructofuranose onto the atomic positions of dideoxyimino-D-glucitol is not possible due to the steric restrictions of the active site. However, by utilizing the approximate 2-fold symmetry of the sugar, a stereochemically sensible model is produced which is consistent with other data. In addition to reaction with alpha-D-furanose, the enzyme probably reacts with open ring keto sugars which are present at significant concentrations. Other sugars which resemble furanoses either do not inhibit significantly or are not observed in the crystals bound in a single conformation. PMID:1457418

  20. Confirmation and Elimination of Xylose Metabolism Bottlenecks in Glucose Phosphoenolpyruvate-Dependent Phosphotransferase System-Deficient Clostridium acetobutylicum for Simultaneous Utilization of Glucose, Xylose, and Arabinose▿†

    PubMed Central

    Xiao, Han; Gu, Yang; Ning, Yuanyuan; Yang, Yunliu; Mitchell, Wilfrid J.; Jiang, Weihong; Yang, Sheng

    2011-01-01

    Efficient cofermentation of d-glucose, d-xylose, and l-arabinose, three major sugars present in lignocellulose, is a fundamental requirement for cost-effective utilization of lignocellulosic biomass. The Gram-positive anaerobic bacterium Clostridium acetobutylicum, known for its excellent capability of producing ABE (acetone, butanol, and ethanol) solvent, is limited in using lignocellulose because of inefficient pentose consumption when fermenting sugar mixtures. To overcome this substrate utilization defect, a predicted glcG gene, encoding enzyme II of the d-glucose phosphoenolpyruvate-dependent phosphotransferase system (PTS), was first disrupted in the ABE-producing model strain Clostridium acetobutylicum ATCC 824, resulting in greatly improved d-xylose and l-arabinose consumption in the presence of d-glucose. Interestingly, despite the loss of GlcG, the resulting mutant strain 824glcG fermented d-glucose as efficiently as did the parent strain. This could be attributed to residual glucose PTS activity, although an increased activity of glucose kinase suggested that non-PTS glucose uptake might also be elevated as a result of glcG disruption. Furthermore, the inherent rate-limiting steps of the d-xylose metabolic pathway were observed prior to the pentose phosphate pathway (PPP) in strain ATCC 824 and then overcome by co-overexpression of the d-xylose proton-symporter (cac1345), d-xylose isomerase (cac2610), and xylulokinase (cac2612). As a result, an engineered strain (824glcG-TBA), obtained by integrating glcG disruption and genetic overexpression of the xylose pathway, was able to efficiently coferment mixtures of d-glucose, d-xylose, and l-arabinose, reaching a 24% higher ABE solvent titer (16.06 g/liter) and a 5% higher yield (0.28 g/g) compared to those of the wild-type strain. This strain will be a promising platform host toward commercial exploitation of lignocellulose to produce solvents and biofuels. PMID:21926197

  1. Production of ethanol from D-xylose by using D-xylose isomerase and yeasts

    SciTech Connect

    Gong, C.S.; Chem, L.F.; Flickinger, M.C.; Chiang, L.C.; Tsao, G.T.

    1981-02-01

    D-xylulose, an intermediate of D-xylose catabolism, was observed to be fermentable to ethanol and carbon dioxide in a yield of greater than 80% by yeasts (including industrial bakers' yeast) under fermentative conditions. This conversion appears to be carried out by many yeasts known for D-glucose fermentation. In some yeasts, xylitol, in addition to ethanol, was produced from D-xylulose. Fermenting yeasts are also able to produce ethanol from D-xylose when D-xylose isomerizing enzyme is present. The results indicate that ethanol could be produced from D-xylose in a yield of greater than 80% by a two-step process. First. D-xylose is converted to D-xylulose by xylose isomerase. D-xylulose is then fermented to ethanol by yeasts.

  2. Formation of xylitol and xylitol-5-phosphate and its impact on growth of d-xylose-utilizing Corynebacterium glutamicum strains.

    PubMed

    Radek, Andreas; Müller, Moritz-Fabian; Gätgens, Jochem; Eggeling, Lothar; Krumbach, Karin; Marienhagen, Jan; Noack, Stephan

    2016-08-10

    Wild-type Corynebacterium glutamicum has no endogenous metabolic activity for utilizing the lignocellulosic pentose d-xylose for cell growth. Therefore, two different engineering approaches have been pursued resulting in platform strains harbouring a functional version of either the Isomerase (ISO) or the Weimberg (WMB) pathway for d-xylose assimilation. In a previous study we found for C. glutamicum WMB by-product formation of xylitol during growth on d-xylose and speculated that the observed lower growth rates are due to the growth inhibiting effect of this compound. Based on a detailed phenotyping of the ISO, WMB and the wild-type strain of C. glutamicum, we here show that this organism has a natural capability to synthesize xylitol from d-xylose under aerobic cultivation conditions. We furthermore observed the intracellular accumulation of xylitol-5-phosphate as a result of the intracellular phosphorylation of xylitol, which was particularly pronounced in the C. glutamicum ISO strain. Interestingly, low amounts of supplemented xylitol strongly inhibit growth of this strain on d-xylose, d-glucose and d-arabitol. These findings demonstrate that xylitol is a suitable substrate of the endogenous xylulokinase (XK, encoded by xylB) and its overexpression in the ISO strain leads to a significant phosphorylation of xylitol in C. glutamicum. Therefore, in order to circumvent cytotoxicity by xylitol-5-phosphate, the WMB pathway represents an interesting alternative route for engineering C. glutamicum towards efficient d-xylose utilization. PMID:27297548

  3. Extracellular Thiol Isomerases and Their Role in Thrombus Formation

    PubMed Central

    Schulman, Sol; Bendapudi, Pavan; Sharda, Anish; Chen, Vivien; Bellido-Martin, Lola; Jasuja, Reema; Furie, Barbara C.; Flaumenhaft, Robert

    2016-01-01

    Abstract Significance: The mammalian endoplasmic reticulum (ER) houses a large family of twenty thioredoxin-like proteins of which protein disulfide isomerase (PDI) is the archetypal member. Although the PDI family is best known for its role in oxidative protein folding of secretory proteins in the ER, these thioredoxin-like proteins fulfill ever-expanding roles, both within the secretory pathway and beyond. Recent Advances: Secreted PDI family proteins have now been shown to serve a critical role in platelet thrombus formation and fibrin generation. Utilizing intravital microscopy to visualize thrombus formation in mice, we have demonstrated the presence of extracellular PDI antigen during thrombus formation following injury of the vascular wall. Inhibition of PDI abrogates thrombus formation in vivo (16, 26, 46, 55). These observations have been extended to other PDI family members, including ERp57 (39, 116, 118, 123) and ERp5 (77). The vascular thiol isomerases are those PDI family members secreted from platelets and/or endothelium (40): PDI, ERp57, ERp5, ERp72, ERp44, ERp29, and TMX3. We focus here on PDI (16, 46, 55), ERp57 (39, 116, 118, 123), and ERp5 (77), which have been implicated in thrombus formation in vivo. Critical Issues: It would appear that a system of thiol isomerase redox catalysts has been hijacked from the ER to regulate thrombus formation in the vasculature. Future Directions: How this redox system is trafficked to and regulated at the cell surface, the identity of extracellular substrates, why so many thiol isomerases are required, and which thiol isomerase functions are necessary are critical unanswered questions in understanding the role of thiol isomerases in thrombus formation. Antioxid. Redox Signal. 24, 1–15. PMID:26467859

  4. Characterization of Triosephosphate Isomerase Mutants with Reduced Enzyme Activity in Mus Musculus

    PubMed Central

    Merkle, S.; Pretsch, W.

    1989-01-01

    Four heterozygous triosephosphate isomerase (TPI) mutants with approximately 50% reduced activity in blood compared to wild type were detected in offspring of 1-ethyl-1-nitrosourea treated male mice. Breeding experiments displayed an autosomal, dominant mode of inheritance for the mutations. All mutations were found to be homozygous lethal at an early postimplantation stage of embryonic development, probably due to a total lack of TPI activity and consequently to the inability to utilize glucose as a source of metabolic energy. Although activity alteration was also found in liver, lung, kidney, spleen, heart, brain and muscle the TPI deficiency in heterozygotes has no influence on the following physiological traits: hematological parameters, plasma glucose, glucose consumption of blood cells, body weight and organo-somatic indices of liver, spleen, heart, kidney and lung. Biochemical investigations of TPI in the four mutant lines indicated no difference of physicochemical properties compared to the wild type. Results from immunoinactivation assays indicate that the decrease of enzyme activity corresponds to a decrease in the level of an immunologically active moiety. It is suggested that the mutations have affected the Tpi-1 structural locus and resulted in alleles which produce no detectable enzyme activity and no immunologically cross-reacting material. The study furthermore suggests one functional TPI gene per haploid genome in the erythrocyte and seven other tested organs of the mouse. PMID:2693209

  5. Protein disulfide isomerase a multifunctional protein with multiple physiological roles

    NASA Astrophysics Data System (ADS)

    Ali Khan, Hyder; Mutus, Bulent

    2014-08-01

    Protein disulfide isomerase (PDI), is a member of the thioredoxin superfamily of redox proteins. PDI has three catalytic activities including, thiol-disulfide oxireductase, disulfide isomerase and redox-dependent chaperone. Originally, PDI was identified in the lumen of the endoplasmic reticulum and subsequently detected at additional locations, such as cell surfaces and the cytosol. This review will provide an overview of the recent advances in relating the structural features of PDI to its multiple catalytic roles as well as its physiological and pathophysiological functions related to redox regulation and protein folding.

  6. Phosphate salts

    MedlinePlus

    ... as a laxative to clean the bowels before surgery or intestinal tests. Healthcare providers sometimes give potassium phosphate intravenously (by IV) for treating low phosphate and high calcium levels in the blood, and for preventing low phosphate in patients who are being tube-fed.

  7. A further polymorphism of the Gd locus for glucose-6-phosphate dehydrogenase present among blacks (Nigerians) and apparently absent among Caucasoids: the quantitative isoelectrophoretic variation of the Gd+ allele.

    PubMed

    Modiano, G; Ciminelli, B M; Malaspina, P; Santolamazza, P; Sedran, L; Meloni, T; Forteleoni, G; Mela, G

    1991-01-01

    A structural but isoelectrophoretic moderate variation of glucose-6-phosphate dehydrogenase (G6PD) activity is common among Nigerians (a black population exposed to a long-lasting intense Plasmodium falciparum malarial endemia). It had never even been searched for among Caucasoids and Mongoloids. In the present work, we attempted to ascertain whether this polymorphism exists among Caucasoids. With this purpose, two Caucasoid male populations were studied: Sardinians and Romans, who respectively did and did not experience an evolutionarily effective exposure to P. falciparum. The approach adopted here consisted in comparing the variations of G6PD activity observed between brothers who certainly received their Gd gene from the same grandparent (hence Gd genes identical by descent) with those between brothers who received it (in the Roman series) or may have received it (in the Sardinian series) from different grandparents. No evidence for common moderate G6PD activity variations segregating with the Gd gene was found either in Romans or Sardinians, who have both been studied with much larger samples and more sensitive approaches than those which detected such type of polymorphism among Nigerians. The upper 95% confidence limit of such zero estimates for the frequency of the isoelectrophoretic quantitative Gd variant alleles were about 0.04 and 0.025 for Romans and Sardinians, respectively. This is the first example of a genetic region (the Gd gene with its flanking sequences) apparently monomorphic in a major race and with several (four) polymorphic sites in another major race. PMID:1797629

  8. 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.

  9. Detailed functional analysis of two clinical glucose-6-phosphate dehydrogenase (G6PD) variants, G6PDViangchan and G6PDViangchan+Mahidol: Decreased stability and catalytic efficiency contribute to the clinical phenotype.

    PubMed

    Boonyuen, Usa; Chamchoy, Kamonwan; Swangsri, Thitiluck; Saralamba, Naowarat; Day, Nicholas P J; Imwong, Mallika

    2016-06-01

    Deficiency of glucose-6-phosphate dehydrogenase (G6PD) is an X-linked hereditary genetic defect that is the most common polymorphism and enzymopathy in humans. To investigate functional properties of two clinical variants, G6PDViangchan and G6PDViangchan+Mahidol, these two mutants were created by overlap-extension PCR, expressed in Escherichia coli and purified to homogeneity. We describe an overexpression and purification method to obtain substantial amounts of functionally active protein. The KM for G6P of the two variants was comparable to the KM of the native enzyme, whereas the KM for NADP(+) was increased 5-fold for G6PDViangchan and 8-fold for G6PDViangchan+Mahidol when compared with the native enzyme. Additionally, kcat of the mutant enzymes was markedly reduced, resulting in a 10- and 18-fold reduction in catalytic efficiency for NADP(+) catalysis for G6PDViangchan and G6PDViangchan+Mahidol, respectively. Furthermore, the two variants demonstrated significant reduction in thermostability, but similar susceptibility to trypsin digestion, when compared with the wild-type enzyme. The presence of NADP(+) is shown to improve the stability of G6PD enzymes. This is the first report indicating that protein instability and reduced catalytic efficiency are responsible for the reduced catalytic activity of G6PDViangchan and G6PDViangchan+Mahidol and, as a consequence, contribute to the clinical phenotypes of these two clinical variants. PMID:27053284

  10. The structure of rhamnose isomerase from Escherichia coli and its relation with xylose isomerase illustrates a change between inter and intra-subunit complementation during evolution.

    PubMed

    Korndörfer, I P; Fessner, W D; Matthews, B W

    2000-07-21

    Using a new expression construct, rhamnose isomerase from Escherichia coli was purified and crystallized. The crystal structure was solved by multiple isomorphous replacement and refined to a crystallographic residual of 17.4 % at 1.6 A resolution. Rhamnose isomerase is a tight tetramer of four (beta/alpha)(8)-barrels. A comparison with other known structures reveals that rhamnose isomerase is most similar to xylose isomerase. Alignment of the sequences of the two enzymes based on their structures reveals a hitherto undetected sequence identity of 13 %, suggesting that the two enzymes evolved from a common precursor. The structure and arrangement of the (beta/alpha)(8)-barrels of rhamnose isomerase are very similar to xylose isomerase. Each enzyme does, however, have additional alpha-helical domains, which are involved in tetramer association, and largely differ in structure. The structures of complexes of rhamnose isomerase with the inhibitor l-rhamnitol and the natural substrate l-rhamnose were determined and suggest that an extended loop, which is disordered in the native enzyme, becomes ordered on substrate binding, and may exclude bulk solvent during catalysis. Unlike xylose isomerase, this loop does not extend across a subunit interface but contributes to the active site of its own subunit. It illustrates how an interconversion between inter and intra-subunit complementation can occur during evolution. In the crystal structure (although not necessarily in vivo) rhamnose isomerase appears to bind Zn(2+) at a "structural" site. In the presence of substrate the enzyme also binds Mn(2+) at a nearby "catalytic" site. An array of hydrophobic residues, not present in xylose isomerase, is likely to be responsible for the recognition of l-rhamnose as a substrate. The available structural data suggest that a metal-mediated hydride-shift mechanism, which is generally favored for xylose isomerase, is also feasible for rhamnose isomerase. PMID:10891278

  11. 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

  12. High-resolution crystal structure and redox properties of chloroplastic triosephosphate isomerase from Chlamydomonas reinhardtii.

    PubMed

    Zaffagnini, Mirko; Michelet, Laure; Sciabolini, Chiara; Di Giacinto, Nastasia; Morisse, Samuel; Marchand, Christophe H; Trost, Paolo; Fermani, Simona; Lemaire, Stéphane D

    2014-01-01

    Triosephosphate isomerase (TPI) catalyzes the interconversion of glyceraldehyde-3-phosphate to dihydroxyacetone phosphate. Photosynthetic organisms generally contain two isoforms of TPI located in both cytoplasm and chloroplasts. While the cytoplasmic TPI is involved in the glycolysis, the chloroplastic isoform participates in the Calvin-Benson cycle, a key photosynthetic process responsible for carbon fixation. Compared with its cytoplasmic counterpart, the functional features of chloroplastic TPI have been poorly investigated and its three-dimensional structure has not been solved. Recently, several studies proposed TPI as a potential target of different redox modifications including dithiol/disulfide interchanges, glutathionylation, and nitrosylation. However, neither the effects on protein activity nor the molecular mechanisms underlying these redox modifications have been investigated. Here, we have produced recombinantly and purified TPI from the unicellular green alga Chlamydomonas reinhardtii (Cr). The biochemical properties of the enzyme were delineated and its crystallographic structure was determined at a resolution of 1.1 Å. CrTPI is a homodimer with subunits containing the typical (β/α)8-barrel fold. Although no evidence for TRX regulation was obtained, CrTPI was found to undergo glutathionylation by oxidized glutathione and trans-nitrosylation by nitrosoglutathione, confirming its sensitivity to multiple redox modifications. PMID:24157611

  13. The role of phosphomannose isomerase in Leishmania mexicana glycoconjugate synthesis and virulence.

    PubMed

    Garami, A; Ilg, T

    2001-03-01

    Phosphomannose isomerase (PMI) catalyzes the reversible interconversion of fructose 6-phosphate and mannose 6-phosphate, which is the first step in the biosynthesis of activated mannose donors required for the biosynthesis of various glycoconjugates. Leishmania species synthesize copious amounts of mannose-containing glycolipids and glycoproteins, which are involved in virulence of these parasitic protozoa. To investigate the role of PMI for parasite glycoconjugate synthesis, we have cloned the PMI gene (lmexpmi) from Leishmania mexicana, generated gene deletion mutants (Delta lmexpmi), and analyzed their phenotype. Delta lmexpmi mutants lack completely the high PMI activity found in wild type parasites, but are, in contrast to fungi, able to grow in media deficient for free mannose. The mutants are unable to synthesize phosphoglycan repeats [-6-Gal beta 1-4Man alpha 1-PO(4)-] and mannose-containing glycoinositolphospholipids, and the surface expression of the glycosylphosphatidylinositol-anchored dominant surface glycoprotein leishmanolysin is strongly decreased, unless the parasite growth medium is supplemented with mannose. The Delta lmexpmi mutant is attenuated in infections of macrophages in vitro and of mice, suggesting that PMI may be a target for anti-Leishmania drug development. L. mexicana Delta lmexpmi provides the first conditional mannose-controlled system for parasite glycoconjugate assembly with potential applications for the investigation of their biosynthesis, intracellular sorting, and function. PMID:11084042

  14. 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...

  15. Identification and analysis of residues contained on β → α loops of the dual-substrate (βα)8 phosphoribosyl isomerase A specific for its phosphoribosyl anthranilate isomerase activity

    PubMed Central

    Noda-García, Lianet; Camacho-Zarco, Aldo R; Verdel-Aranda, Karina; Wright, Helena; Soberón, Xavier; Fülöp, Vilmos; Barona-Gómez, Francisco

    2010-01-01

    A good model to experimentally explore evolutionary hypothesis related to enzyme function is the ancient-like dual-substrate (βα)8 phosphoribosyl isomerase A (PriA), which takes part in both histidine and tryptophan biosynthesis in Streptomyces coelicolor and related organisms. In this study, we determined the Michaelis–Menten enzyme kinetics for both isomerase activities in wild-type PriA from S. coelicolor and in selected single-residue monofunctional mutants, identified after Escherichia coli in vivo complementation experiments. Structural and functional analyses of a hitherto unnoticed residue contained on the functionally important β → α loop 5, namely, Arg139, which was postulated on structural grounds to be important for the dual-substrate specificity of PriA, is presented for the first time. Indeed, enzyme kinetics analyses done on the mutant variants PriA_Ser81Thr and PriA_Arg139Asn showed that these residues, which are contained on β → α loops and in close proximity to the N-terminal phosphate-binding site, are essential solely for the phosphoribosyl anthranilate isomerase activity of PriA. Moreover, analysis of the X-ray crystallographic structure of PriA_Arg139Asn elucidated at 1.95 Å herein strongly implicates the occurrence of conformational changes in this β → α loop as a major structural feature related to the evolution of the dual-substrate specificity of PriA. It is suggested that PriA has evolved by tuning a fine energetic balance that allows the sufficient degree of structural flexibility needed for accommodating two topologically dissimilar substrates—within a bifunctional and thus highly constrained active site—without compromising its structural stability. PMID:20066665

  16. Evidence for distinct dehydrogenase and isomerase sites within a single 3. beta. -hydroxysteroid dehydrogenase/5-ene-4-ene isomerase protein

    SciTech Connect

    Luu-The, V.; Takahashi, Masakazu; de Launoit, Y.; Dumont, M.; Lachance, Y.; Labrie, F. )

    1991-09-10

    Complementary DNA encoding human 3{beta}-hydroxysteroid dehydrogenase/5-ene-4-ene isomerase (3-{beta}-HSD) has been expressed in transfected GH{sub 4}C{sub 1} with use of the cytomegalovirus promoter. The activity of the expressed protein clearly shows that both dehydrogenase and isomerase enzymatic activities are present within a single protein. However, such findings do not indicate whether the two activities reside within one or two closely related catalytic sites. With use of ({sup 3}H)-5-androstenedione, the intermediate compound in dehydroepiandrosterone (DHEA) transformation into 4-androstenedione by 3{beta}-HSD, the present study shows that 4MA (N,N-diethyl-4-methyl-3-oxo-4-aza-5{alpha}-androstane-17{beta}-carboxamide) and its analogues of 5-androstenedione to 4-androstenedione with an approximately 1,000-fold higher K{sub i} value. The present results thus strongly suggest that dehydrogenase and isomerase activities are present at separate sites on the 3-{beta}-HSD protein. Such data suggest that the irreversible step in the transformation of DHEA to 4-androstenedione is due to a separate site possessing isomerase activity that converts the 5-ene-3-keto to a much more stable 4-ene-3-keto configuration.

  17. Protein disulfide isomerase ameliorates β-cell dysfunction in pancreatic islets overexpressing human islet amyloid polypeptide.

    PubMed

    Montane, Joel; de Pablo, Sara; Obach, Mercè; Cadavez, Lisa; Castaño, Carlos; Alcarraz-Vizán, Gema; Visa, Montserrat; Rodríguez-Comas, Júlia; Parrizas, Marcelina; Servitja, Joan Marc; Novials, Anna

    2016-01-15

    Human islet amyloid polypeptide (hIAPP) is the major component of amyloid deposits in islets of type 2 diabetic patients. hIAPP misfolding and aggregation is one of the factors that may lead to β-cell dysfunction and death. Endogenous chaperones are described to be important for the folding and functioning of proteins. Here, we examine the effect of the endoplasmic reticulum chaperone protein disulfide isomerase (PDI) on β-cell dysfunction. Among other chaperones, PDI was found to interact with hIAPP in human islet lysates. Furthermore, intrinsically recovered PDI levels were able to restore the effect of high glucose- and palmitate-induced β-cell dysfunction by increasing 3.9-fold the glucose-stimulated insulin secretion levels and restoring insulin content up to basal control values. Additionally, PDI transduction decreased induced apoptosis by glucolipotoxic conditions. This approach could reveal a new therapeutic target and aid in the development of strategies to improve β-cell dysfunction in type 2 diabetic patients. PMID:26607804

  18. A paradigm for enzyme-catalyzed proton transfer at carbon: triosephosphate isomerase.

    PubMed

    Richard, John P

    2012-04-01

    Triosephosphate isomerase (TIM) catalyzes the stereospecific 1,2-proton shift at dihydroxyacetone phosphate (DHAP) to give (R)-glyceraldehyde 3-phosphate through a pair of isomeric enzyme-bound cis-enediolate phosphate intermediates. The chemical transformations that occur at the active site of TIM were well understood by the early 1990s. The mechanism for enzyme-catalyzed isomerization is similar to that for the nonenzymatic reaction in water, but the origin of the catalytic rate acceleration is not understood. We review the results of experimental work that show that a substantial fraction of the large 12 kcal/mol intrinsic binding energy of the nonreacting phosphodianion fragment of TIM is utilized to activate the active site side chains for catalysis of proton transfer. Evidence is presented that this activation is due to a phosphodianion-driven conformational change, the most dramatic feature of which is closure of loop 6 over the dianion. The kinetic data are interpreted within the framework of a model in which activation is due to the stabilization by the phosphodianion of a rare, desolvated, loop-closed form of TIM. The dianion binding energy is proposed to drive the otherwise thermodynamically unfavorable desolvation of the solvent-exposed active site. This reduces the effective local dielectric constant of the active site, to enhance stabilizing electrostatic interactions between polar groups and the anionic transition state, and increases the basicity of the carboxylate side chain of Glu-165 that functions to deprotonate the bound carbon acid substrate. A rebuttal is presented to the recent proposal [Samanta, M., Murthy, M. R. N., Balaram, H., and Balaram, P. (2011) ChemBioChem 12, 1886-1895] that the cationic side chain of K12 functions as an active site electrophile to protonate the carbonyl oxygen of DHAP. PMID:22409228

  19. Solubilization of insoluble inorganic phosphate by Burkholderia cepacia DA23 isolated from cultivated soil

    PubMed Central

    Song, Ok-Ryul; Lee, Seung-Jin; Lee, Yong-Seok; Lee, Sang-Cheol; Kim, Keun-Ki; Choi, Yong-Lark

    2008-01-01

    A mineral phosphate solubilizing bacterium, Burkholderia cepacia DA23 has been isolated from cultivated soils. Phosphate-solubilizing activities of the strain against three types of insoluble phosphate were quantitatively determined. When 3% of glucose concentration was used for carbon source, the strain had a marked mineral phosphate-solubilizing activity. Mineral phosphate solubilization was directly related to the pH drop by the strain. Analysis of the culture medium by high pressure liquid chromatography identified gluconic acid as the main organic acid released by Burkholderia cepacia DA23. Gluconic acid production was apparently the result of the glucose dehydrogenase activity and glucose dehydrogenase was affected by phosphate regulation. PMID:24031195

  20. UDP-glucose is a potential intracellular signal molecule in the control of expression of sigma S and sigma S-dependent genes in Escherichia coli.

    PubMed Central

    Böhringer, J; Fischer, D; Mosler, G; Hengge-Aronis, R

    1995-01-01

    The sigma S subunit of RNA polymerase is the master regulator of a regulatory network that controls stationary-phase induction as well as osmotic regulation of many genes in Escherichia coli. In an attempt to identify additional regulatory components in this network, we have isolated Tn10 insertion mutations that in trans alter the expression of osmY and other sigma S-dependent genes. One of these mutations conferred glucose sensitivity and was localized in pgi (encoding phosphoglucose isomerase). pgi::Tn10 strains exhibit increased basal levels of expression of osmY and otsBA in exponentially growing cells and reduced osmotic inducibility of these genes. A similar phenotype was also observed for pgm and galU mutants, which are deficient in phosphoglucomutase and UDP-glucose pyrophosphorylase, respectively. This indicates that the observed effects on gene expression are related to the lack of UDP-glucose (or a derivative thereof), which is common to all three mutants. Mutants deficient in UDP-galactose epimerase (galE mutants) and trehalose-6-phosphate synthase (otsA mutants) do not exhibit such an effect on gene expression, and an mdoA mutant that is deficient in the first step of the synthesis of membrane-derived oligosaccharides, shows only a partial increase in the expression of osmY. We therefore propose that the cellular content of UDP-glucose serves as an internal signal that controls expression of osmY and other sigma S-dependent genes. In addition, we demonstrate that pgi, pgm, and galU mutants contain increased levels of sigma S during steady-state growth, indicating that UDP-glucose interferes with the expression of sigma S itself. PMID:7814331

  1. Interference by acetaminophen in the glucose oxidase-peroxidase method for blood glucose determination.

    PubMed

    Kaufmann-Raab, I; Jonen, H G; Jähnchen, E; Kahl, G F; Groth, U

    1976-10-01

    Acetaminophen, p-aminophenol, and oxyphenbutazone interfere with the glucose oxidase/peroxidase method for glucose. Structurally related compounds that lack a free phenolic hydroxyl group (acetanilide, aniline, and phenylbutazone) do not interfere. During the analytical procedure acetaminophen is consumed. One mole of acetaminophen leads to an apparent loss of four moles of glucose. The hexokinase/glucose-6-phosphate dehydrogenase method (Boehringer Hexokinase method) is not affected by these substances. PMID:975521

  2. Exploring the chemistry and evolution of the isomerases

    PubMed Central

    2016-01-01

    Isomerization reactions are fundamental in biology, and isomers usually differ in their biological role and pharmacological effects. In this study, we have cataloged the isomerization reactions known to occur in biology using a combination of manual and computational approaches. This method provides a robust basis for comparison and clustering of the reactions into classes. Comparing our results with the Enzyme Commission (EC) classification, the standard approach to represent enzyme function on the basis of the overall chemistry of the catalyzed reaction, expands our understanding of the biochemistry of isomerization. The grouping of reactions involving stereoisomerism is straightforward with two distinct types (racemases/epimerases and cis-trans isomerases), but reactions entailing structural isomerism are diverse and challenging to classify using a hierarchical approach. This study provides an overview of which isomerases occur in nature, how we should describe and classify them, and their diversity. PMID:26842835

  3. Exploring the chemistry and evolution of the isomerases.

    PubMed

    Martínez Cuesta, Sergio; Rahman, Syed Asad; Thornton, Janet M

    2016-02-16

    Isomerization reactions are fundamental in biology, and isomers usually differ in their biological role and pharmacological effects. In this study, we have cataloged the isomerization reactions known to occur in biology using a combination of manual and computational approaches. This method provides a robust basis for comparison and clustering of the reactions into classes. Comparing our results with the Enzyme Commission (EC) classification, the standard approach to represent enzyme function on the basis of the overall chemistry of the catalyzed reaction, expands our understanding of the biochemistry of isomerization. The grouping of reactions involving stereoisomerism is straightforward with two distinct types (racemases/epimerases and cis-trans isomerases), but reactions entailing structural isomerism are diverse and challenging to classify using a hierarchical approach. This study provides an overview of which isomerases occur in nature, how we should describe and classify them, and their diversity. PMID:26842835

  4. Methods of measuring Protein Disulfide Isomerase activity: a critical overview

    NASA Astrophysics Data System (ADS)

    Watanabe, Monica; Laurindo, Francisco; Fernandes, Denise

    2014-09-01

    Protein disulfide isomerase is an essential redox chaperone from the endoplasmic reticulum (ER) and is responsible for correct disulfide bond formation in nascent proteins. PDI is also found in other cellular locations in the cell, particularly the cell surface. Overall, PDI contributes to ER and global cell redox homeostasis and signaling. The knowledge about PDI structure and function progressed substantially based on in vitro studies using recombinant PDI and chimeric proteins. In these experimental scenarios, PDI reductase and chaperone activities are readily approachable. In contrast, assays to measure PDI isomerase activity, the hallmark of PDI family, are more complex. Assessment of PDI roles in cells and tissues mainly relies on gain- or loss-of-function studies. However, there is limited information regarding correlation of experimental readouts with the distinct types of PDI activities. In this mini-review, we evaluate the main methods described for measuring the different kinds of PDI activity: thiol reductase, thiol oxidase, thiol isomerase and chaperone. We emphasize the need to use appropriate controls and the role of critical interferents (e.g., detergent, presence of reducing agents). We also discuss the translation of results from in vitro studies with purified recombinant PDI to cellular and tissue samples, with critical comments on the interpretation of results.

  5. Binding Energy and Catalysis by D-Xylose Isomerase: Kinetic, Product and X-Ray Crystallographic Analysis of Enzyme-Catalyzed Isomerization of (R)-Glyceraldehyde‡, ¶

    PubMed Central

    Toteva, Maria M.; Silvaggi, Nicholas R.; Allen, Karen N.; Richard, John P.

    2011-01-01

    D-Xylose isomerase (XI) and triosephosphate isomerase (TIM) catalyze the aldose-ketose isomerization reactions of D-xylose and D-glyceraldehyde 3-phosphate (DGAP), respectively. D-Glyceraldehyde (DGA) is the triose fragment common to the substrates for XI and TIM. The XI-catalyzed isomerization of DGA to give dihydroxyacetone (DHA) in D2O was monitored by 1H NMR spectroscopy and kcat/Km = 0.034 M−1 s−1 was determined for this isomerization at pD 7.0. This is similar to kcat/Km = 0.017 M−1 s−1 for the TIM-catalyzed carbon deprotonation reaction of DGA in D2O at pD 7.0 [Amyes, T. L.; O’Donoghue, A. C. and Richard J. P. (2001) J. Am. Chem. Soc. 123, 11325–11326]. The much larger activation barrier for XI-catalyzed isomerization of D-xylose (kcat/Km = 490 M−1 s−1) than for the TIM-catalyzed isomerization of DGAP (kcat/Km = 9.6 x 106 M−1 s−1) is due to: (i) The larger barrier to conversion of cyclic D-xylose to the reactive linear sugar (5.4 kcal/mol) than for conversion of DGAP hydrate to the free aldehyde (1.7 kcal/mol). (ii) The smaller intrinsic binding energy [Jencks, W. P. (1975) Adv. Enzymol. Relat. Areas Mol. Biol. 43, 219–410] of the terminal ethylene glycol fragment of D-xylose (9.3 kcal/mol) than of the phosphodianion group of DGAP (ca. 12 kcal/mol). The XI-catalyzed isomerization of DGA in D2O at pD 7.0 gives a 90% yield of [1-1H]-DHA and a 10% yield of [1-2H]-DHA, the product of isomerization with deuterium incorporation from solvent D2O. By comparison, the transfer of 3H from labeled hexose substrate to solvent is observed only once in every 109 turnovers for the XI-catalyzed isomerization of [2-3H]-glucose in H2O [Allen, K. N., Lavie, A., Farber, G. K., Glasfeld, A., Petsko, G. A., and Ringe, D. (1994), Biochemistry 33, 1481–1487]. We propose that truncation of the terminal ethylene glycol fragment of D-xylose to give DGA results in a large decrease in the rate of XI-catalyzed isomerization with hydride transfer compared with that

  6. Glucose-6-Phosphate Dehydrogenase Deficiency Overview

    MedlinePlus

    ... and researchers. OMIM is maintained by Johns Hopkins University School of Medicine. Orphanet is a European reference ... Support for Patients and Families Help with Travel Costs How to Get Involved in Research FAQs About ...

  7. Glucose-6-Phosphate Dehydrogenase Deficiency Overview

    MedlinePlus

    ... gov to read descriptions of these studies. Organizations Organizations Listen Nonprofit support and advocacy groups bring together ... endorsement by GARD. Suggest an organization to add. Organizations Supporting this Disease The G6PD Favism Association E- ...

  8. A method for the production of D-tagatose using a recombinant Pichia pastoris strain secreting β-D-galactosidase from Arthrobacter chlorophenolicus and a recombinant L-arabinose isomerase from Arthrobacter sp. 22c

    PubMed Central

    2012-01-01

    Background D-Tagatose is a natural monosaccharide which can be used as a low-calorie sugar substitute in food, beverages and pharmaceutical products. It is also currently being tested as an anti-diabetic and obesity control drug. D-Tagatose is a rare sugar, but it can be manufactured by the chemical or enzymatic isomerization of D-galactose obtained by a β-D-galactosidase-catalyzed hydrolysis of milk sugar lactose and the separation of D-glucose and D-galactose. L-Arabinose isomerases catalyze in vitro the conversion of D-galactose to D-tagatose and are the most promising enzymes for the large-scale production of D-tagatose. Results In this study, the araA gene from psychrotolerant Antarctic bacterium Arthrobacter sp. 22c was isolated, cloned and expressed in Escherichia coli. The active form of recombinant Arthrobacter sp. 22c L-arabinose isomerase consists of six subunits with a combined molecular weight of approximately 335 kDa. The maximum activity of this enzyme towards D-galactose was determined as occurring at 52°C; however, it exhibited over 60% of maximum activity at 30°C. The recombinant Arthrobacter sp. 22c L-arabinose isomerase was optimally active at a broad pH range of 5 to 9. This enzyme is not dependent on divalent metal ions, since it was only marginally activated by Mg2+, Mn2+ or Ca2+ and slightly inhibited by Co2+ or Ni2+. The bioconversion yield of D-galactose to D-tagatose by the purified L-arabinose isomerase reached 30% after 36 h at 50°C. In this study, a recombinant Pichia pastoris yeast strain secreting β-D-galactosidase Arthrobacter chlorophenolicus was also constructed. During cultivation of this strain in a whey permeate, lactose was hydrolyzed and D-glucose was metabolized, whereas D-galactose was accumulated in the medium. Moreover, cultivation of the P. pastoris strain secreting β-D-galactosidase in a whey permeate supplemented with Arthrobacter sp. 22c L-arabinose isomerase resulted in a 90% yield of lactose hydrolysis, the

  9. Radiation inactivation analysis of rat liver microsomal glucose 6-phosphatase

    SciTech Connect

    Ness, G.C.; Sample, C.E.; McCreery, M.J.; Sukalski, K.A.; Nordlie, R.C.

    1986-05-01

    Attempts to obtain the molecular weight of microsomal glucose-6-phosphatase based on solubilization and purification have yielded widely divergent results. Since radiation inactivation analysis can be used to obtain molecular weights of proteins within the native membrane environments, this technique was applied. Identical target sizes of about 70 kd for both glucose 6-phosphate phosphohydrolase and carbamyl phosphate:glucose phosphotransferase were observed. This value was unaffected by adding deoxycholate, which disrupts the microsomal membranes, to the microsomal suspensions prior to irradiation. The data suggest that the glucose 6-phosphate transport function and the glucose 6-phosphate phosphohydrolase activity of microsomal glucose 6-phosphatase either residue on a single polypeptide or on two covalently linked polypeptides.

  10. Risks of Hemolysis in Glucose-6-Phosphate Dehydrogenase Deficient Infants Exposed to Chlorproguanil-Dapsone, Mefloquine and Sulfadoxine-Pyrimethamine as Part of Intermittent Presumptive Treatment of Malaria in Infants

    PubMed Central

    Poirot, Eugenie; Vittinghoff, Eric; Ishengoma, Deus; Alifrangis, Michael; Carneiro, Ilona; Hashim, Ramadhan; Baraka, Vito; Mosha, Jacklin; Gesase, Samwel; Chandramohan, Daniel; Gosling, Roland

    2015-01-01

    Background Chlorproguanil-dapsone (CD) has been linked to hemolysis in symptomatic glucose-6-phosphate dehydrogenase deficient (G6PDd) children. Few studies have explored the effects of G6PD status on hemolysis in children treated with Intermittent Preventive Treatment in infants (IPTi) antimalarial regimens. We sought to examine the joint effects of G6PD status and IPTi antimalarial treatment on incidence of hemolysis in asymptomatic children treated with CD, sulfadoxine-pyrimethamine (SP), and mefloquine (MQ). Methods A secondary analysis of data from a double-blind, placebo-controlled trial of IPTi was conducted. Hemoglobin (Hb) measurements were made at IPTi doses, regular follow-up and emergency visits. G6PD genotype was determined at 9 months looking for SNPs for the A- genotype at coding position 202. Multivariable linear and logistic regression models were used to examine hemolysis among children with valid G6PD genotyping results. Hemolysis was defined as the absolute change in Hb or as any post-dose Hb <8 g/dL. These outcomes were assessed using either a single follow-up Hb on day 7 after an IPTi dose or Hb obtained 1 to 14 or 28 days after each IPTi dose. Findings Relative to placebo, CD reduced Hb by approximately 0.5 g/dL at day 7 and within 14 days of an IPTi dose, and by 0.2 g/dL within 28 days. Adjusted declines in the CD group were larger than in the MQ and SP groups. At day 7, homo-/hemizygous genotype was associated with higher odds of Hb <8 g/dL (adjusted odds ratio = 6.7, 95% CI 1.7 to 27.0) and greater absolute reductions in Hb (-0.6 g/dL, 95% CI -1.1 to 0.003). There was no evidence to suggest increased reductions in Hb among homo-/hemizygous children treated with CD compared to placebo, SP or MQ. Conclusions While treatment with CD demonstrated greater reductions in Hb at 7 and 14 days after an IPTi dose compared to both SP and MQ, there was no evidence that G6PD deficiency exacerbated the adverse effects of CD, despite evidence for higher

  11. [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

  12. 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

  13. Phosphate–Induced Renal Fibrosis Requires the Prolyl Isomerase Pin1

    PubMed Central

    Shiizaki, Kazuhiro; Kuro-o, Makoto; Malter, James S.

    2016-01-01

    Tubulo-interstitial fibrosis is a common, destructive endpoint for a variety of kidney diseases. Fibrosis is well correlated with the loss of kidney function in both humans and rodents. The identification of modulators of fibrosis could provide novel therapeutic approaches to reducing disease progression or severity. Here, we show that the peptidyl-prolyl isomerase Pin1 is an important molecular contributor that facilitates renal fibrosis in a well-characterized animal model. While wild-type mice fed a high phosphate diet (HPD) for 8–12 weeks developed calcium deposition, macrophage infiltration and extracellular matrix (ECM) accumulation in the kidney interstitium, Pin1 null mice showed significantly less pathology. The serum Pi in both WT and KO mice were significantly increased by the HPD, but the serum Ca was slightly decreased in KO compared to WT. In addition, both WT and KO HPD mice had less weight gain but exhibited normal organ mass (kidney, lung, spleen, liver and heart). Unexpectedly, renal function was not initially impaired in either genotype irrespective of the HPD. Our results suggest that diet containing high Pi induces rapid renal fibrosis before a significant impact on renal function and that Pin1 plays an important role in the fibrotic process. PMID:26914452

  14. Disruption of PHO13 improves ethanol production via the xylose isomerase pathway.

    PubMed

    Bamba, Takahiro; Hasunuma, Tomohisa; Kondo, Akihiko

    2016-03-01

    Xylose is the second most abundant sugar in lignocellulosic materials and can be converted to ethanol by recombinant Saccharomyces cerevisiae yeast strains expressing heterologous genes involved in xylose assimilation pathways. Recent research demonstrated that disruption of the alkaline phosphatase gene, PHO13, enhances ethanol production from xylose by a strain expressing the xylose reductase (XR) and xylitol dehydrogenase (XDH) genes; however, the yield of ethanol is poor. In this study, PHO13 was disrupted in a recombinant strain harboring multiple copies of the xylose isomerase (XI) gene derived from Orpinomyces sp., coupled with overexpression of the endogenous xylulokinase (XK) gene and disruption of GRE3, which encodes aldose reductase. The resulting YΔGP/XK/XI strain consumed 2.08 g/L/h of xylose and produced 0.88 g/L/h of volumetric ethanol, for an 86.8 % theoretical ethanol yield, and only YΔGP/XK/XI demonstrated increase in cell concentration. Transcriptome analysis indicated that expression of genes involved in the pentose phosphate pathway (GND1, SOL3, TAL1, RKI1, and TKL1) and TCA cycle and respiratory chain (NDE1, ACO1, ACO2, SDH2, IDH1, IDH2, ATP7, ATP19, SDH4, SDH3, CMC2, and ATP15) was upregulated in the YΔGP/XK/XI strain. And the expression levels of 125 cell cycle genes were changed by deletion of PHO13. PMID:26769491

  15. Inhibition of d-xylose isomerase by polyols: atomic details by joint X-ray/neutron crystallography

    SciTech Connect

    Kovalevsky, Andrey; Hanson, B. Leif; Mason, Sax A.; Forsyth, V. Trevor; Fisher, Zoe; Mustyakimov, Marat; Blakeley, Matthew P.; Keen, David A.; Langan, Paul

    2012-09-01

    A joint X-ray/neutron structure of d-xylose isomerase in complex with the inhibitor sorbitol was determined at room temperature at an acidic pH of 5.9. Protonation of the O5 O atom of the sugar was directly observed in the nuclear density maps. Under acidic conditions sorbitol gains a water-mediated interaction with the enzyme active site, which may explain the increased potency of the inhibitor at low pH. d-Xylose isomerase (XI) converts the aldo-sugars xylose and glucose to their keto analogs xylulose and fructose, but is strongly inhibited by the polyols xylitol and sorbitol, especially at acidic pH. In order to understand the atomic details of polyol binding to the XI active site, a 2.0 Å resolution room-temperature joint X-ray/neutron structure of XI in complex with Ni{sup 2+} cofactors and sorbitol inhibitor at pH 5.9 and a room-temperature X-ray structure of XI containing Mg{sup 2+} ions and xylitol at the physiological pH of 7.7 were obtained. The protonation of oxygen O5 of the inhibitor, which was found to be deprotonated and negatively charged in previous structures of XI complexed with linear glucose and xylulose, was directly observed. The Ni{sup 2+} ions occupying the catalytic metal site (M2) were found at two locations, while Mg{sup 2+} in M2 is very mobile and has a high B factor. Under acidic conditions sorbitol gains a water-mediated interaction that connects its O1 hydroxyl to Asp257. This contact is not found in structures at basic pH. The new interaction that is formed may improve the binding of the inhibitor, providing an explanation for the increased affinity of the polyols for XI at low pH.

  16. Cryptococcal phosphoglucose isomerase is required for virulence factor production, cell wall integrity and stress resistance.

    PubMed

    Zhang, Ping; Wei, Dongsheng; Li, Zhongming; Sun, Zhixiong; Pan, Jiao; Zhu, Xudong

    2015-11-01

    Regulation of virulence factor production in the pathogen Cryptococcus neoformans remains to be fully illustrated. We present here a finding that a gene, encoding the glycolysis enzyme phosphoglucose isomerase (Pgi1), is critical for the biosynthesis of melanin and capsule, cell wall integrity and resistance to stress conditions. A leaky mutant of the yeast, LZM19, resulted from an insertion of T-DNA in the PGI1 promoter region, expressed PGI1 at a level only 1.9% of the wild type. LZM19 could synthesize the pigment melanin in the presence of 2% glucose, suggesting a status of LAC1 derepression. Phenotypically, capsule biosynthesis in LZM19 was remarkably reduced. Integrity of the cell wall and plasma membrane of LZM19 were impaired based on its sensitivity to Congo red and SDS. Also, LZM19 exhibited hypersensitivity to osmotic stress generated by 2 M NaCl or 1 M KCl, indicating possible impairment in the HOG signaling pathway. Furthermore, LZM19 failed to utilize mannose and fructose, suggesting a possible involvement of Pgi1 in the breakdown of these two sugars. Our results revealed a crucial role of PGI1 in coordination of the production of virulence factors, cell wall integrity and stress response in C. neoformans. PMID:26271120

  17. Glucose metabolism and effect of acetate in ovine adipocytes.

    PubMed

    Yang, Y T; White, L S; Muir, L A

    1982-08-01

    Isolated ovine adipocytes were incubated in vitro with specifically labeled 14C-glucose in the presence or absence of acetate. The flux patterns of glucose carbon through major metabolic pathways were estimated. When glucose was added as the sole substrate, approximately equal portions of glucose carbon (10%) were oxidized to CO2 in the pentose phosphate pathway, in the pyruvate dehydrogenase reaction and in the citrate cycle. Fifteen percent of the glucose carbon was incorporated into fatty acids and 43% was released as lactate and pyruvate. Addition of acetate to the medium increased glucose carbon uptake by 1.5-fold. Most of this increase was accounted for by a sevenfold increase in the activity of the pentose phosphate pathway. Acetate increased glucose carbon fluxes via pentose phosphate pathway to triose phosphates, from triose phosphate to pyruvate, into glyceride glycerol, into lactate and pyruvate and into pyruvate dehydrogenase and citrate cycle CO2. Glucose carbon incorporated into fatty acids was decreased 50% by acetate while, carbon fluxes through the phosphofructokinase-aldolase reactions were not significantly increased. Results of this study suggest that, when glucose is the sole substrate, the conversion of glucose to fatty acids in ovine adipocytes may not be limited by the maximum capacity of hexokinase, the pentose phosphate pathway or enzymes involved in the conversion of triose phosphates to pyruvate and of pyruvate to fatty acid. Acetate increased glucose utilization apparently by increasing activity of the pentose phosphate pathway as a result of enhanced NADPH utilization for fatty acid synthesis. PMID:7142048

  18. Dexamethasone increases glucose cycling, but not glucose production, in healthy subjects

    SciTech Connect

    Wajngot, A.; Khan, A.; Giacca, A.; Vranic, M.; Efendic, S. )

    1990-11-01

    We established that measurement of glucose fluxes through glucose-6-phosphatase (G-6-Pase; hepatic total glucose output, HTGO), glucose cycling (GC), and glucose production (HGP), reveals early diabetogenic changes in liver metabolism. To elucidate the mechanism of the diabetogenic effect of glucocorticoids, we treated eight healthy subjects with oral dexamethasone (DEX; 15 mg over 48 h) and measured HTGO with (2-3H)glucose and HGP with (6-3H)glucose postabsorptively and during a 2-h glucose infusion (11.1 mumol.kg-1.min-1). (2-3H)- minus (6-3H)glucose equals GC. DEX significantly increased plasma glucose, insulin, C peptide, and HTGO, while HGP was unchanged. In controls and DEX, glucose infusion suppressed HTGO (82 vs. 78%) and HGP (87 vs. 91%). DEX increased GC postabsorptively (three-fold) P less than 0.005 and during glucose infusion (P less than 0.05) but decreased metabolic clearance and glucose uptake (Rd), which eventually normalized, however. Because DEX increased HTGO (G-6-Pase) and not HGP (glycogenolysis + gluconeogenesis), we assume that DEX increases HTGO and GC in humans by activating G-6-Pase directly, rather than by expanding the glucose 6-phosphate pool. Hyperglycemia caused by peripheral effects of DEX can also contribute to an increase in GC by activating glucokinase. Therefore, measurement of glucose fluxes through G-6-Pase and GC revealed significant early effects of DEX on hepatic glucose metabolism, which are not yet reflected in HGP.

  19. Metabolomic and 13C-Metabolic Flux Analysis of a Xylose-Consuming Saccharomyces cerevisiae Strain Expressing Xylose Isomerase

    PubMed Central

    Wasylenko, Thomas M.; Stephanopoulos, Gregory

    2016-01-01

    Over the past two decades significant progress has been made in the engineering of xylose-consuming Saccharomyces cerevisiae strains for production of lignocellulosic biofuels. However, the ethanol productivities achieved on xylose are still significantly lower than those observed on glucose for reasons that are not well understood. We have undertaken an analysis of central carbon metabolite pool sizes and metabolic fluxes on glucose and on xylose under aerobic and anaerobic conditions in a strain capable of rapid xylose assimilation via xylose isomerase in order to investigate factors that may limit the rate of xylose fermentation. We find that during xylose utilization the flux through the non-oxidative PPP is high but the flux through the oxidative PPP is low, highlighting an advantage of the strain employed in this study. Furthermore, xylose fails to elicit the full carbon catabolite repression response that is characteristic of glucose fermentation in S. cerevisiae. We present indirect evidence that the incomplete activation of the fermentation program on xylose results in a bottleneck in lower glycolysis, leading to inefficient re-oxidation of NADH produced in glycolysis. PMID:25311863

  20. Diversity of Specificity and Function of Phosphate Translocators in Various Plastids

    PubMed Central

    Heldt, Hans W.; Flügge, Ulf-Ingo; Borchert, Sieglinde

    1991-01-01

    This report gives a comparison of the specificity of phosphate translocators in various plastids. Whereas the phosphate translocator of the C3 plant spinach mediates a counter exchange between inorganic phosphate, dihydroxyacetone phosphate, and 3-phosphoglycerate, the phosphate translocators in chloroplasts from C4 and CAM plants transport phosphoenolpyruvate in addition to the above mentioned metabolites. In plastids from pea roots the phosphate translocator also transports glucose 6-phosphate. This diversity of phosphate translocators is discussed in view of the special functions of the various plastids. PMID:16667988

  1. Protein disulfide isomerase mediates glutathione depletion-induced cytotoxicity.

    PubMed

    Okada, Kazushi; Fukui, Masayuki; Zhu, Bao-Ting

    2016-08-26

    Glutathione depletion is a distinct cause underlying many forms of pathogenesis associated with oxidative stress and cytotoxicity. Earlier studies showed that glutamate-induced glutathione depletion in immortalized murine HT22 hippocampal neuronal cells leads to accumulation of reactive oxygen species (ROS) and ultimately cell death, but the precise mechanism underlying these processes is not clear. Here we show that during the induction of glutathione depletion, nitric oxide (NO) accumulation precedes ROS accumulation. While neuronal NO synthase (nNOS) in untreated HT22 cells exists mostly as a monomer, glutathione depletion results in increased formation of the dimer nNOS, accompanied by increases in the catalytic activity. We identified that nNOS dimerization is catalyzed by protein disulfide isomerase (PDI). Inhibition of PDI's isomerase activity effectively abrogates glutathione depletion-induced conversion of monomer nNOS into dimer nNOS, accumulation of NO and ROS, and cytotoxicity. Furthermore, we found that PDI is present in untreated cells in an inactive S-nitrosylated form, which becomes activated following glutathione depletion via S-denitrosylation. These results reveal a novel role for PDI in mediating glutathione depletion-induced oxidative cytotoxicity, as well as its role as a valuable therapeutic target for protection against oxidative cytotoxicity. PMID:27317486

  2. Converting a Sulfenic Acid Reductase into a Disulfide Bond Isomerase

    PubMed Central

    Chatelle, Claire; Kraemer, Stéphanie; Ren, Guoping; Chmura, Hannah; Marechal, Nils; Boyd, Dana; Roggemans, Caroline; Ke, Na; Riggs, Paul; Bardwell, James

    2015-01-01

    Abstract Aims: Posttranslational formation of disulfide bonds is essential for the folding of many secreted proteins. Formation of disulfide bonds in a protein with more than two cysteines is inherently fraught with error and can result in incorrect disulfide bond pairing and, consequently, misfolded protein. Protein disulfide bond isomerases, such as DsbC of Escherichia coli, can recognize mis-oxidized proteins and shuffle the disulfide bonds of the substrate protein into their native folded state. Results: We have developed a simple blue/white screen that can detect disulfide bond isomerization in vivo, using a mutant alkaline phosphatase (PhoA*) in E. coli. We utilized this screen to isolate mutants of the sulfenic acid reductase (DsbG) that allowed this protein to act as a disulfide bond isomerase. Characterization of the isolated mutants in vivo and in vitro allowed us to identify key amino acid residues responsible for oxidoreductase properties of thioredoxin-like proteins such as DsbC or DsbG. Innovation and Conclusions: Using these key residues, we also identified and characterized interesting environmental homologs of DsbG with novel properties, thus demonstrating the capacity of this screen to discover and elucidate mechanistic details of in vivo disulfide bond isomerization. Antioxid. Redox Signal. 23, 945–957. PMID:26191605

  3. Sequence of the Ampullariella sp. strain 3876 gene coding for xylose isomerase.

    PubMed Central

    Saari, G C; Kumar, A A; Kawasaki, G H; Insley, M Y; O'Hara, P J

    1987-01-01

    The nucleotide sequence of the gene coding for xylose isomerase from Ampullariella sp. strain 3876, a gram-positive bacterium, has been determined. A clone of a fragment of strain 3876 DNA coding for a xylose isomerase activity was identified by its ability to complement a xylose isomerase-defective Escherichia coli strain. One such complementation positive fragment, 2,922 nucleotides in length, was sequenced in its entirety. There are two open reading frames 1,182 and 1,242 nucleotides in length, on opposite strands of this fragment, each of which could code for a protein the expected size of xylose isomerase. The 1,182-nucleotide open reading frame was identified as the coding sequence for the protein from the sequence analysis of the amino-terminal region and selected internal peptides. The gene initiates with GTG and has a high guanine and cytosine content (70%) and an exceptionally strong preference (97%) for guanine or cytosine in the third position of the codons. The gene codes for a 43,210-dalton polypeptide composed of 393 amino acids. The xylose isomerase from Ampullariella sp. strain 3876 is similar in size to other bacterial xylose isomerases and has limited amino acid sequence homology to the available sequences from E. coli, Bacillus subtilis, and Streptomyces violaceus-ruber. In all cases yet studied, the bacterial gene for xylulose kinase is downstream from the gene for xylose isomerase. We present evidence suggesting that in Ampullariella sp. strain 3876 these genes are similarly arranged. PMID:3027039

  4. Kinetics and specificity of homogeneous protein disulphide-isomerase in protein disulphide isomerization and in thiol-protein-disulphide oxidoreduction.

    PubMed

    Lambert, N; Freedman, R B

    1983-07-01

    The protein disulphide-bond isomerization activity of highly active homogeneous protein disulphide-isomerase (measured by re-activation of 'scrambled' ribonuclease) is enhanced by EDTA and by phosphate buffers. As shown for previous less-active preparations, the enzyme has a narrow pH optimum around pH 7.8 and requires the presence of either a dithiol or a thiol. The dithiol dithiothreitol is effective at concentrations 100-fold lower than the monothiols reduced glutathione and cysteamine. The enzyme follows Michaelis-Menten kinetics with respect to these substrates; Km values are 4,620 and 380 microM respectively. The enzyme shows apparent inhibition by high concentrations of thiol or dithiol compounds (greater than 10 X Km), but the effect is mainly on the extent of reaction, not the initial rate. This is interpreted as indicating the formation of significant amounts of reduced ribonuclease in these more reducing conditions. The purified enzyme will also catalyse net reduction of insulin disulphide bonds by reduced glutathione (i.e. it has thiol:protein-disulphide oxidoreductase or glutathione:insulin transhydrogenase activity), but this requires considerably higher concentrations of enzyme and reduced glutathione than does the disulphide-isomerization activity. The Km for reduced glutathione in this reaction is an order of magnitude greater than that for the disulphide-isomerization activity, and the turnover number is considerably lower than that of other enzymes that can catalyse thiol-disulphide oxidoreduction. Conventional two-substrate steady-state analysis of the thiol:protein-disulphide oxidoreductase activity indicates that it follows a ternary-complex mechanism. The protein disulphide-isomerase and thiol:protein-disulphide oxidoreductase activities co-purify quantitatively through the final stages of purification, implying that a single protein species is responsible for both activities. It is concluded that previous preparations, from various sources

  5. Oxygen transfer as a tool for fine-tuning recombinant protein production by Pichia pastoris under glyceraldehyde-3-phosphate dehydrogenase promoter.

    PubMed

    Güneş, Hande; Çalık, Pınar

    2016-07-01

    Effects of oxygen transfer on recombinant protein production by Pichia pastoris under glyceraldehyde-3-phosphate dehydrogenase promoter were investigated. Recombinant glucose isomerase was chosen as the model protein. Two groups of oxygen transfer strategies were applied, one of which was based on constant oxygen transfer rate where aeration rate was Q O/V = 3 and 10 vvm, and agitation rate was N = 900 min(-1); while the other one was based on constant dissolved oxygen concentrations, C DO = 5, 10, 15, 20 and 40 % in the fermentation broth, by using predetermined exponential glucose feeding with μ o = 0.15 h(-1). The highest cell concentration was obtained as 44 g L(-1) at t = 9 h of the glucose fed-batch phase at C DO = 20 % operation while the highest volumetric and specific enzyme activities were obtained as 4440 U L(-1) and 126 U g(-1) cell, respectively at C DO = 15 % operation. Investigation of specific enzyme activities revealed that keeping C DO at 15 % was more advantageous with an expense of relatively higher by-product formation and lower specific cell growth rate. For this strategy, the highest oxygen transfer coefficient and oxygen uptake rate were K L a = 0.045 s(-1) and OUR = 8.91 mmol m(-3) s(-1), respectively. PMID:27003824

  6. Plant tissue hybrid electrode for determination of phosphate and fluoride

    SciTech Connect

    Schubert, F.; Renneberg, R.; Scheller, F.W.; Kirstein, L.

    1984-08-01

    A biosensor for inorganic phosphate and fluoride has been developed by coupling a potato (Solanum tuberosum) tissue slice and immobilized glucose oxidase with a Clark oxygen electrode. Measurement is based on the inhibition by either ion of potato acid phosphates catalyzed glucose 6-phosphate hydrolysis. The precision is 1.7% and 6.5% and the lower detection limit 2.5 X 10/sup -5/ M and 1 X 10/sup -4/ M for phosphate and fluoride, respectively. For phosphate determination the hybrid sensor is stable for 28 days or 300 assays. With a higher limit of detection the sensor can be applied in a commercial enzyme electrode based device. Its application for phosphate determination in fertilizer and urine samples is described.

  7. [Phosphate solubilization of Aureobasidium pullulan F4 and its mechanism].

    PubMed

    Wang, Dan; Zhan, Jing; Sun, Qing-Ye

    2014-07-01

    The Aureobasidium pullulans F4 was isolated from the rhizosphere of Hippochaete ramosissimum in Tongguanshan mine wasteland in Tongling City, Anhui Province. Liquid culture was conducted with four kinds of phosphorus sources, calcium phosphate, aluminum phosphate, ferric phosphate and rock phosphate to determine the pH, dissolved phosphorus, phosphorus in the bacteria and organic acid in the solution. The results showed that the phosphate solubilization by A. pullulans F4 varied with phosphorus sources, which decreased in order of aluminum phosphate > ferric phosphate, calcium phosphate > rock phosphate. The amounts of dissolved phosphorus in the different treatments were all higher than 200 mg x L(-1). The pH of the medium dropped immediately in 48 h, and the aluminum phosphate and ferric phosphate treatments showed a greater decrease in pH than the calcium phosphate and rock phosphate treatments. The organic acid synthesized by A. pullulans F4 included oxalic acid, citric acid and tartaric acid, and oxalic acid, among which oxalic acid was the dominated component. The phosphate dissolving capacity of A. pullulans F4 showed no significant correlation with organic acid, but significantly correlated with the pH. The available phosphorus was significantly improved with the combined application of A. pullulans F4 and glucose, suggesting A. pullulans F4 was a potent candidate for remediation of copper mine wastelands. PMID:25345061

  8. Glucose metabolism in rat retinal pigment epithelium.

    PubMed

    Coffe, Víctor; Carbajal, Raymundo C; Salceda, Rocío

    2006-01-01

    The retinal pigment epithelium (RPE) is the major transport pathway for exchange of metabolites and ions between choroidal blood supply and the neural retina. To gain insight into the mechanisms controlling glucose metabolism in RPE and its possible relationship to retinopathy, we studied the influence of different glucose concentrations on glycogen and lactate levels and CO(2) production in RPE from normal and streptozotocin-treated diabetic rats. Incubation of normal RPE in the absence of glucose caused a decrease in lactate production and glycogen content. In normal RPE, increasing glucose concentrations from 5.6 mM to 30 mM caused a four-fold increase in glucose accumulation and CO(2) yield, as well as reduction in lactate and glycogen production. In RPE from diabetic rats glucose accumulation did not increase in the presence of high glucose substrate, but it showed a four- and a seven-fold increase in CO(2) production through the mitochondrial and pentose phosphate pathways, respectively. We found high glycogen levels in RPE which can be used as an energy reserve for RPE itself and/or neural retina. Findings further show that the RPE possesses a high oxidative capacity. The large increase in glucose shunting to the pentose phosphate pathway in diabetic retina exposed to high glucose suggests a need for reducing capacity, consistent with increased oxidative stress. PMID:16475003

  9. BIOLOGICAL ELECTRODES SPECIFIC FOR PHOSPHATE AND NITRITE IONS

    EPA Science Inventory

    Biological electrodes have been developed for the selective analysis of phosphate and nitrate ions at micro-levels. A phosphate electrode was constructed by using two enzymes, alkaline phosphatase and glucose oxidase which were immobilized and mounted on the surface of the Platin...

  10. Isolation, purification, and characterization of thermophilic T80 isoenzyme of xylose isomerase from the xerophyte Cereus pterogonus.

    PubMed

    Ravikumar, Sambandam; Shyamala, Sivalingam; Muthuraman, Pandurangan; Srikumar, Kotteazeth

    2011-01-01

    A thermostable isoenzyme (T(80)) of xylose isomerase from the eukaryote xerophyte Cereus pterogonus was purified to homogeneity by precipitation with ammonium sulfate and column chromatography on Dowex-1 ion exchange, with Sephadex G-100 gel filtration, resulting in an approximately 25.55-fold increase in specific activity and a final yield of approximately 17.9%. Certain physiochemical and kinetic properties (K(m) and V(max)) of the T(80) xylose isomerase isoenzyme were investigated. The molecular mass of the purified T(80) isoenzyme was 68 kD determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Polyclonal antibodies against the purified T(80) isoenzyme recognized a single polypeptide band on Western blots. The activation energy required for the thermal denaturation of the isoenzyme was determined to be 61.84 KJ mol(-1). The use of differential scanning calorimetry established the melting temperature of the CPXI isoenzyme to be 80°C, but when studied with added metal ions, melting temperature increases to more than the normal. Fluorescence spectroscopy of T(80) isoenzymes yielded an emission peak with λ(em) at 320 nm and 340 nm, respectively, confirming the presence of Trp residue in these proteins. Electron paramagnetic resonance (EPR) analysis at liquid nitrogen temperature established the presence of Mn(2+) and Co(2+) associated with each isoenzyme. These enzyme species exhibited different thermal and pH stabilities compared to their mesophilic counterparts and offered greater efficiency in functioning as a potential alternate catalytic converter of glucose in the production of high-fructose corn syrup (HFCS) for the sweetener industry and for ethanol production. PMID:21442549

  11. Absence of phosphoglucose isomerase-1 in retinal photoreceptor, pigment epithelium and Muller cells.

    PubMed

    Archer, Simon N; Ahuja, Poonam; Caffé, Romeo; Mikol, Catherine; Foster, Russell G; van Veen, Theo; von Schantz, Malcolm

    2004-06-01

    Macroarray analysis was used to compare equal amounts of cDNA from wild-type and rd/rd (retinal degeneration) mice, collected at P90 when photoreceptor degeneration is virtually complete. A stronger signal for the glycolytic enzyme phosphoglucose isomerase (Gpi1) was observed in the rd/rd sample. Extracellularly, Gpi1 may act as a cytokine, independently described as neuroleukin and autocrine motility factor. Retinal Gpi1 expression was investigated by Northern and Western blot analysis and immunohistochemistry. Double-labelling was performed with antibodies against Gpi1 and calbindin-D, glutamine synthetase, RPE65, calretinin and ultraviolet opsin in order to provide positive cell type identification. Northern and Western blots showed double expression levels per microgram of RNA and protein, respectively, in the rd/rd retina compared with wild-type. However, the total amount of Gpi1 protein per retina was indistinguishable. Gpi1 immunoreactivity was found in ganglion, amacrine, horizontal and bipolar cells, but not in rods, cones, pigment epithelium and Muller cells. This distribution explains why the absolute amounts of Gpi1 protein were not appreciably different between wild-type and the rd/rd phenotype, where rods and cones are absent, whilst the relative contribution of Gpi1 to the total protein and RNA pools differed. Some extracellular immunoreactivity was observed in the photoreceptor matrix around cones in freshly fixed tissue only, which could possibly reflect a role as a cytokine. We propose that glycolysis in Gpi1-negative cells proceeds entirely through the pentose phosphate pathway, creating NADPH at the cost of organic carbon. We hypothesize that the unique metabolic needs of photoreceptors justify this trade-off. PMID:15182299

  12. Biochemical properties of duplicated isozymes of phosphoglucose isomerase in the plant Clarkia xantiana.

    PubMed

    Gottlieb, L D; Greve, L C

    1981-02-01

    The structural gene locus specifying subunits of cytoplasmic isoenzymes of phosphoglucose isomerase (PGI) is present in duplicate in many diploid species of Clarkia (Onagraceae), a genus of annual plants native to California. We studied the kinetic properties and molecular weights of a large number of genetically defined and highly purified PGIs in C. xantiana, a number of species with the duplication, as a means of examining the biochemical consequences of the evolution of a new gene locus. This species is primarily outcrossing, but also includes several previously described predominantly self-pollinating populations. Both cytoplasmic PGI loci in the outcrossing populations are polymorphic and their enzyme products are readily separated by electrophoresis. The PGIs from the outcrossing populations were rated by electrophoresis. The PGIs from the outcrossing populations were generally closely similar in molecular weight, pH optimum, heat sensitivity, energy activation, and apparent Km (fructose-6-phosphate). The PGI loci in the selfing populations are monomorphic and specify enzymes having identical electrophoretic mobilities to those coded by the most frequent alleles of the outcrosser. The PGI isozymes in the selfers differed fivefold in Km, suggesting that they have a very different catalytic effectiveness. The high Km of the PGI-3A' isozyme (1.1mM) was anomalous among the PGIs examined and would likely be disadvantageous in a species which lacked other more normally functioning PGIs. But in the cytoplasm of the selfing plants, it is present with other PGIs that have low Km values. The PGI-3A' enzyme is a good candidate for a gene product coded by a "forbidden" mutation that could not have been established in the absence of the duplication. The rationale for this suggestion is described and it is also pointed out that the divergence of duplicated genes is influenced by many factors such as the breeding system and other population factors as well as the effect of

  13. Regulation of isopropylmalate isomerase synthesis in Neurospora crassa.

    PubMed Central

    Reichenbecher, V E; Fischer, M; Gross, S R

    1978-01-01

    The capacity to synthetize isopropylmalate isomerase (EC 4.2.1.33) by Neurospora crassa increased during induction in the presence of cycloheximide but was inhibited by proflavine and other inhibitors of RNA synthesis. Turnover of the enzyme once formed appeared negligible, but the message (measured as enzyme-forming capacity) had a half-life of 4 to 8 min. A comparison of the kinetics of induction in the wild type and a newly isolated alpha-isopropylmalate-permeable strain suggested strongly that feedback control by leucine of alpha-isopropylmalate production can adequately serve as the primary physiological regulator of endogenous inducer concentration. Genetic data are presented which implicate the involvement of two unlinked genes, ipm-1 and ipm-2, in determining permeation of alpha-isopropylmalate. PMID:146702

  14. Mammalian peptide isomerase: platypus-type activity is present in mouse heart.

    PubMed

    Koh, Jennifer M S; Chow, Stephanie J P; Crossett, Ben; Kuchel, Philip W

    2010-06-01

    Male platypus (Ornithorhynchus anatinus) venom has a peptidyl aminoacyl L/D-isomerase (hereafter called peptide isomerase) that converts the second amino acid residue in from the N-terminus from the L- to the D-form, and vice versa. A reversed-phase high-performance liquid chromatography (RP-HPLC) assay has been developed to monitor the interconversion using synthetic hexapeptides derived from defensin-like peptide-2 (DLP-2) and DLP-4 as substrates. It was hypothesised that animals other than the platypus would have peptide isomerase with the same substrate specificity. Accordingly, eight mouse tissues were tested and heart was shown to have the activity. This is notable for being the first evidence of a peptide isomerase being present in a higher mammal and heralds finding the activity in man. PMID:20564672

  15. Structural features of normal and complemented forms of the Neurospora isopropylmalate isomerase.

    PubMed Central

    Reichenbecher, V E; Gross, S R

    1978-01-01

    The isopropylmalate isomerase (EC 4.2.1.33) of Neurospora crassa is a globular protein consisting of a single polypeptide chain with a molecular weight of about 90,000. The isomerase cannot easily be freed of a contaminating protease which cleaves the enzyme into two major fragments, one of approximately 56,000 and the other 37,000 daltons. This suggests that the folded polypeptide chain may contain some hinge point or loop exposed on the surface which makes it susceptible to proteolytic attack. Most of the isomerase activity extracted from the wild-type strain is in monomer form. However, a small fraction of the activity in crude extracts is found in multimeric aggregates, and the active isomerase extracted from complementing leu-2 heterokaryons consists entirely of dimers and higher multimers. These observations suggest that, though active as a monomer, a significant fraction of the normal enzyme might be organized in multimeric form within the cell. Images PMID:146703

  16. Trichinella spiralis: genome database searches for the presence and immunolocalization of protein disulphide isomerase family members.

    PubMed

    Freitas, C P; Clemente, I; Mendes, T; Novo, C

    2016-01-01

    The formation of nurse cells in host muscle cells during Trichinella spiralis infection is a key step in the infective mechanism. Collagen trimerization is set up via disulphide bond formation, catalysed by protein disulphide isomerase (PDI). In T. spiralis, some PDI family members have been identified but no localization is described and no antibodies specific for T. spiralis PDIs are available. In this work, computational approaches were used to search for non-described PDIs in the T. spiralis genome database and to check the cross-reactivity of commercial anti-human antibodies with T. spiralis orthologues. In addition to a previously described PDI (PDIA2), endoplasmic reticulum protein (ERp57/PDIA3), ERp72/PDIA4, and the molecular chaperones calreticulin (CRT), calnexin (CNX) and immunoglobulin-binding protein/glucose-regulated protein (BIP/GRP78), we identified orthologues of the human thioredoxin-related-transmembrane proteins (TMX1, TMX2 and TMX3) in the genome protein database, as well as ERp44 (PDIA10) and endoplasmic reticulum disulphide reductase (ERdj5/PDIA19). Immunocytochemical staining of paraffin sections of muscle infected by T. spiralis enabled us to localize some orthologues of the human PDIs (PDIA3 and TMX1) and the chaperone GRP78. A theoretical three-dimensional model for T. spiralis PDIA3 was constructed. The localization and characteristics of the predicted linear B-cell epitopes and amino acid sequence of the immunogens used for commercial production of anti-human PDIA3 antibodies validated the use of these antibodies for the immunolocalization of T. spiralis PDIA3 orthologues. These results suggest that further study of the role of the PDIs and chaperones during nurse cell formation is desirable. PMID:25475092

  17. Detection of platypus-type L/D-peptide isomerase activity in aqueous extracts of papaya fruit.

    PubMed

    Arakawa, Kensuke; Koh, Jennifer M S; Crossett, Ben; Torres, Allan M; Kuchel, Philip W

    2012-09-01

    Peptide isomerase catalyses the post-translational isomerisation of the L: - to the D: -form of an amino acid residue around the N/C-termini of substrate peptides. To date, some peptide isomerases have been found in a limited number of animal secretions and cells. We show here that papaya extracts have weak peptide isomerase activity. The activity was detected in each 30-100 kDa fraction of the flesh and the seed extracts of unripe and ripe papaya fruit. The definitive activity was confirmed in the ripe papaya extracts, but even then it was much less active than that of the other peptide isomerases previously reported. The activity was markedly inhibited by methanol, and partly so by amastatin and diethyl pyrocarbonate. This is the first report of peptide isomerase activity in a plant and suggests that perhaps every living organism may have some peptide isomerase activity. PMID:22648682

  18. 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

  19. Glucose Variability

    PubMed Central

    2013-01-01

    The proposed contribution of glucose variability to the development of the complications of diabetes beyond that of glycemic exposure is supported by reports that oxidative stress, the putative mediator of such complications, is greater for intermittent as opposed to sustained hyperglycemia. Variability of glycemia in ambulatory conditions defined as the deviation from steady state is a phenomenon of normal physiology. Comprehensive recording of glycemia is required for the generation of any measurement of glucose variability. To avoid distortion of variability to that of glycemic exposure, its calculation should be devoid of a time component. PMID:23613565

  20. Linoleic acid isomerase from Propionibacterium acnes: purification, characterization, molecular cloning, and heterologous expression.

    PubMed

    Deng, Ming-De; Grund, Alan D; Schneider, Kenneth J; Langley, Kim M; Wassink, Sarah L; Peng, Susan S; Rosson, Reinhardt A

    2007-12-01

    Propionibacterium acnes strain ATCC 6919 catalyzes the isomerization of the double bond at the C9 position in linoleic acid (c9,c12, 18:2) to form t10,c12 conjugated linoleic acid (CLA, 18:2). CLA has significant health benefits in animal and human. The linoleic acid C9 isomerase was purified to an apparent homogeneity by successive chromatography on diethylaminoethyl (DEAE) anion exchange, hydrophobic interaction, and chromatofocusing columns. Two degenerated oligonucleotide primers were synthesized according to the N-terminal peptide sequence to clone, by polymerase chain reaction (PCR), a short nucleotide sequence (62 bp) of the isomerase gene. The linoleic acid isomerase gene (lai) was subsequently cloned by inverse PCR. The amino acid sequence deduced from the lai coding sequence predicts a protein of 424 amino acid residues (48 kDa), excluding the N-terminal methionine, which was absent in the polypeptide purified from the native host. The isomerase shares no significant sequence homology to other enzymes except a flavin-binding domain in the N-terminal region. The recombinant isomerase purified from Escherichia coli showed a typical ultraviolet spectrum for FAD-bound proteins. The recombinant enzyme produced a single isomer of t10,c12-CLA from linoleic acid, as demonstrated by gas chromatography and gas chromatography-mass spectrum analysis. The recombinant isomerase protein was expressed at high levels in E. coli, but it was almost totally sequestered in inclusion bodies. The level of active isomerase was increased 376-fold by medium and process optimization in bench-scale fermentors. PMID:18057448

  1. 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.

  2. Genetic and functional aspects of linoleate isomerase in Lactobacillus acidophilus.

    PubMed

    Macouzet, Martin; Robert, Normand; Lee, Byong H

    2010-08-01

    While the remarkable health effects of conjugated linoleic acid (CLA) catalyzed from alpha-linoleic acid by the enzyme linoleate isomerase (LI, EC 5.2.1.5) are well recognized, how widely this biochemical activity is present and the mechanisms of its regulation in lactic acid bacteria are unknown. Although certain strains of Lactobacillus acidophilus can enrich CLA in fermented dairy products, it is unknown if other strains share this capacity. Due to its immense economic importance, this work aimed to investigate genetic aspects of CLA production in L. acidophilus for the first time. The genomic DNA from industrial and type strains of L. acidophilus were subjected to PCR and immunoblot analyses using the putative LI gene of L. reuteri ATCC 55739 as probe. The CLA production ability was estimated by gas chromatography of the biomass extracts. The presumptive LI gene from L. acidophilus ATCC 832 was isolated and sequenced. The resulting sequence shared 71% identity with that of L. reuteri and at least 99% with reported sequences from other L. acidophilus strains. All the strains accumulated detectable levels of CLA and tested positive by PCR and immunoblotting. However, no apparent correlation was observed between the yields and the hybridization patterns. The results suggest that LI activity might be common among L. acidophilus and related species and provide a new tool for screening potential CLA producers. PMID:20461509

  3. Identification of disulfide bond isomerase substrates reveals bacterial virulence factors

    PubMed Central

    Ren, Guoping; Champion, Matthew M.; Huntley, Jason F.

    2014-01-01

    Summary Bacterial pathogens are exposed to toxic molecules inside the host and require efficient systems to form and maintain correct disulfide bonds for protein stability and function. The intracellular pathogen Francisella tularensis encodes a disulfide bond formation protein ortholog, DsbA, which previously was reported to be required for infection of macrophages and mice. However, the molecular mechanisms by which F. tularensis DsbA contributes to virulence are unknown. Here, we demonstrate that F. tularensis DsbA is a bifunctional protein that oxidizes and, more importantly, isomerizes complex disulfide connectivity in substrates. A single amino acid in the conserved cis-proline loop of the DsbA thioredoxin domain was shown to modulate both isomerase activity and F. tularensis virulence. Trapping experiments in F. tularensis identified over 50 F. tularensis DsbA substrates, including outer membrane proteins, virulence factors, and many hypothetical proteins. Six of these hypothetical proteins were randomly selected and deleted, revealing two novel proteins, FTL_1548 and FTL_1709, which are required for F. tularensis virulence. We propose that the extreme virulence of F. tularensis is partially due to the bifunctional nature of DsbA, that many of the newly-identified substrates are required for virulence, and that the development of future DsbA inhibitors could have broad anti-bacterial implications. PMID:25257164

  4. The Oligomeric Nature of Triosephosphate Isomerase. Studies of Monomerization

    NASA Astrophysics Data System (ADS)

    Zárate-Pérez, Francisco; Vázquez-Contreras, Edgar

    2008-02-01

    In this work, we report the implications of the monomerization of triosephosphate isomerase (TIM) from T. cruzi (TcTIM). A monomeric mutant (monoTcTIM) of this species was constructed by genetic engineering, shortening the main loop of interdigitation, which is fundamental for its dimerization. The properties of monoTcTIM were compared with those of the other TIM monomeric versions: genetically engineered mutants, or equilibrium intermediates obtained by chemical denaturation. The stability for almost all the monomeric variants so far reported appears in the same range, and also presents similar structural characteristics. Regarding the catalytic activity of monomeric mutants, when it is present is in several orders of magnitude lower than those observed in their respective wild-type enzyme. The change in the hydrophobic surface of TcTIM after and before monomerization was also studied and corresponds to an extensive area of the interface region, which becomes exposed when monomers are dissociated. This fact could be related with conformational changes in the local environment of the catalytic amino acids responsible for the isomerization of the substrate and consequently the inactivation of its catalytic properties. Comparison of the structural, folding and unfolding properties, as well as stability studies could give answers on why this enzyme is an obligate oligomer.

  5. Purification and Partial characterization of Trypanosoma cruzi triosephosphate isomerase.

    PubMed

    Bourguignon, S C; Meirelles, M N; Pacheco, R S; De Simone, S G

    1998-01-01

    The enzyme triosephosphate isomerase (TPI, EC 5.3.1.1) was purified from extracts of epimastigote forms of Trypanosoma cruzi. The purification steps included: hydrophobic interaction chromatography on phenyl-Sepharose, CM-Sepharose, and high performance liquid gel filtration chromatography. The CM-Sepharose material contained two bands (27 and 25 kDa) with similar isoelectric points (pI 9.3-9.5) which could be separated by gel filtration in high performance liquid chromatography. Polyclonal antibodies raised against the porcine TPI detected one single polypeptide on western blot with a molecular weight (27 kDa) identical to that purified from T. cruzi. These antibodies also recognized only one band of identical molecular weight in western blots of several other trypanosomatids (Blastocrithidia culicis, Crithidia desouzai, Phytomonas serpens, Herpertomonas samuelpessoai). The presence of only one enzymatic form of TPI in T. cruzi epimastigotes was confirmed by agarose gel activity assay and its localization was established by immunocytochemical analysis. The T. cruzi purified TPI (as well as other trypanosomatid' TPIs) is a dimeric protein, composed of two identical subunits with an approximate mw of 27,000 and it is resolved on two dimensional gel electrophoresis with a pI of 9.3. Sequence analysis of the N-terminal portion of the 27 kDa protein revealed a high homology to Leishmania mexicana and T. brucei proteins. PMID:9698898

  6. Catalytic mechanism of a retinoid isomerase essential for vertebrate vision

    DOE PAGESBeta

    Kiser, Philip D.; Zhang, Jianye; Badiee, Mohsen; Li, Qingjiang; Shi, Wuxian; Sui, Xuewu; Golczak, Marcin; Tochtrop, Gregory P.; Palczewski, Krzysztof

    2015-04-20

    Visual function in vertebrates is dependent on the membrane-bound retinoid isomerase RPE65, an essential component of the retinoid cycle pathway that regenerates 11-cis-retinal for rod and cone opsins. The mechanism by which RPE65 catalyzes stereoselective retinoid isomerization has remained elusive because of uncertainty about how retinoids bind to its active site. Here we present crystal structures of RPE65 in complex with retinoid-mimetic compounds, one of which is in clinical trials for the treatment of age-related macular degeneration. The structures reveal the active site retinoid-binding cavity located near the membrane-interacting surface of the enzyme as well as an Fe-bound palmitate ligandmore » positioned in an adjacent pocket. With the geometry of the RPE65–substrate complex clarified, we delineate a mechanism of catalysis that reconciles the extensive biochemical and structural research on this enzyme. Finally, these data provide molecular foundations for understanding a key process in vision and pharmacological inhibition of RPE65 with small molecules.« less

  7. Catalytic mechanism of a retinoid isomerase essential for vertebrate vision

    SciTech Connect

    Kiser, Philip D.; Zhang, Jianye; Badiee, Mohsen; Li, Qingjiang; Shi, Wuxian; Sui, Xuewu; Golczak, Marcin; Tochtrop, Gregory P.; Palczewski, Krzysztof

    2015-04-20

    Visual function in vertebrates is dependent on the membrane-bound retinoid isomerase RPE65, an essential component of the retinoid cycle pathway that regenerates 11-cis-retinal for rod and cone opsins. The mechanism by which RPE65 catalyzes stereoselective retinoid isomerization has remained elusive because of uncertainty about how retinoids bind to its active site. Here we present crystal structures of RPE65 in complex with retinoid-mimetic compounds, one of which is in clinical trials for the treatment of age-related macular degeneration. The structures reveal the active site retinoid-binding cavity located near the membrane-interacting surface of the enzyme as well as an Fe-bound palmitate ligand positioned in an adjacent pocket. With the geometry of the RPE65–substrate complex clarified, we delineate a mechanism of catalysis that reconciles the extensive biochemical and structural research on this enzyme. Finally, these data provide molecular foundations for understanding a key process in vision and pharmacological inhibition of RPE65 with small molecules.

  8. ALS-linked protein disulfide isomerase variants cause motor dysfunction.

    PubMed

    Woehlbier, Ute; Colombo, Alicia; Saaranen, Mirva J; Pérez, Viviana; Ojeda, Jorge; Bustos, Fernando J; Andreu, Catherine I; Torres, Mauricio; Valenzuela, Vicente; Medinas, Danilo B; Rozas, Pablo; Vidal, Rene L; Lopez-Gonzalez, Rodrigo; Salameh, Johnny; Fernandez-Collemann, Sara; Muñoz, Natalia; Matus, Soledad; Armisen, Ricardo; Sagredo, Alfredo; Palma, Karina; Irrazabal, Thergiory; Almeida, Sandra; Gonzalez-Perez, Paloma; Campero, Mario; Gao, Fen-Biao; Henny, Pablo; van Zundert, Brigitte; Ruddock, Lloyd W; Concha, Miguel L; Henriquez, Juan P; Brown, Robert H; Hetz, Claudio

    2016-04-15

    Disturbance of endoplasmic reticulum (ER) proteostasis is a common feature of amyotrophic lateral sclerosis (ALS). Protein disulfide isomerases (PDIs) areERfoldases identified as possibleALSbiomarkers, as well as neuroprotective factors. However, no functional studies have addressed their impact on the disease process. Here, we functionally characterized fourALS-linked mutations recently identified in two majorPDIgenes,PDIA1 andPDIA3/ERp57. Phenotypic screening in zebrafish revealed that the expression of thesePDIvariants induce motor defects associated with a disruption of motoneuron connectivity. Similarly, the expression of mutantPDIs impaired dendritic outgrowth in motoneuron cell culture models. Cellular and biochemical studies identified distinct molecular defects underlying the pathogenicity of thesePDImutants. Finally, targetingERp57 in the nervous system led to severe motor dysfunction in mice associated with a loss of neuromuscular synapses. This study identifiesERproteostasis imbalance as a risk factor forALS, driving initial stages of the disease. PMID:26869642

  9. Catalytic mechanism of a retinoid isomerase essential for vertebrate vision

    PubMed Central

    Kiser, Philip D.; Zhang, Jianye; Badiee, Mohsen; Li, Qingjiang; Shi, Wuxian; Sui, Xuewu; Golczak, Marcin; Tochtrop, Gregory P.; Palczewski, Krzysztof

    2015-01-01

    Visual function in vertebrates is dependent on the membrane-bound retinoid isomerase, RPE65, an essential component of the retinoid cycle pathway that regenerates 11-cis-retinal for rod and cone opsins. The mechanism by which RPE65 catalyzes stereoselective retinoid isomerization has remained elusive due to uncertainty about how retinoids bind to its active site. Here we present crystal structures of RPE65 in complex with retinoid-mimetic compounds, one of which is in clinical trials for treatment of age-related macular degeneration. The structures reveal the active site retinoid-binding cavity located near the membrane-interacting surface of the enzyme as well as an Fe-bound palmitate ligand positioned in an adjacent pocket. With the geometry of the RPE65-substrate complex clarified we delineate a mechanism of catalysis that reconciles the extensive biochemical and structural research on this enzyme. These data provide molecular foundations for understanding a key process in vision and pharmacological inhibition of RPE65 with small molecules. PMID:25894083

  10. Plant phosphomannose isomerase as a selectable marker for rice transformation

    PubMed Central

    Hu, Lei; Li, Hao; Qin, Ruiying; Xu, Rongfang; Li, Juan; Li, Li; Wei, Pengcheng; Yang, Jianbo

    2016-01-01

    The E. coli phosphomannose isomerase (EcPMI) gene is widely used as a selectable marker gene (SMG) in mannose (Man) selection-based plant transformation. Although some plant species exhibit significant PMI activity and active PMIs were even identified in Man-sensitive plants, whether plant PMIs can be used as SMGs remains unclear. In this study, we isolated four novel PMI genes from Chlorella variabilis and Oryza sativa. Their isoenzymatic activities were examined in vitro and compared with that of EcPMI. The active plant PMIs were separately constructed into binary vectors as SMGs and then transformed into rice via Agrobacterium. In both Indica and Japonica subspecies, our results indicated that the plant PMIs could select and produce transgenic plants in a pattern similar to that of EcPMI. The transgenic plants exhibited an accumulation of plant PMI transcripts and enhancement of the in vivo PMI activity. Furthermore, a gene of interest was successfully transformed into rice using the plant PMIs as SMGs. Thus, novel SMGs for Man selection were isolated from plants, and our analysis suggested that PMIs encoding active enzymes might be common in plants and could potentially be used as appropriate genetic elements in cisgenesis engineering. PMID:27174847

  11. Roles of Prolyl Isomerases in RNA-Mediated Gene Expression

    PubMed Central

    Thapar, Roopa

    2015-01-01

    The peptidyl-prolyl cis-trans isomerases (PPIases) that include immunophilins (cyclophilins and FKBPs) and parvulins (Pin1, Par14, Par17) participate in cell signaling, transcription, pre-mRNA processing and mRNA decay. The human genome encodes 19 cyclophilins, 18 FKBPs and three parvulins. Immunophilins are receptors for the immunosuppressive drugs cyclosporin A, FK506, and rapamycin that are used in organ transplantation. Pin1 has also been targeted in the treatment of Alzheimer’s disease, asthma, and a number of cancers. While these PPIases are characterized as molecular chaperones, they also act in a nonchaperone manner to promote protein-protein interactions using surfaces outside their active sites. The immunosuppressive drugs act by a gain-of-function mechanism by promoting protein-protein interactions in vivo. Several immunophilins have been identified as components of the spliceosome and are essential for alternative splicing. Pin1 plays roles in transcription and RNA processing by catalyzing conformational changes in the RNA Pol II C-terminal domain. Pin1 also binds several RNA binding proteins such as AUF1, KSRP, HuR, and SLBP that regulate mRNA decay by remodeling mRNP complexes. The functions of ribonucleoprotein associated PPIases are largely unknown. This review highlights PPIases that play roles in RNA-mediated gene expression, providing insight into their structures, functions and mechanisms of action in mRNP remodeling in vivo. PMID:25992900

  12. Purification and characterization of corticosteroid side chain isomerase

    SciTech Connect

    Marandici, A.; Monder, C. )

    1990-02-06

    Corticosteroid side chain isomerase of rat liver catalyzes the interconversion of the ketol (20-oxo-21-ol) and (20-hydroxy-21-al) forms of the corticosteroid side chain. The enzyme has now been purified to apparent homogeneity from rat liver cytosol by sequential chromatography on anionic, hydroxylapatite, and gel filtration columns. Ketol-aldol isomerization is followed by measuring the exchange of tritium from 21-tritiated steroids with water. The native enzyme is a dimer of MW 44,000. The isoelectric point is 4.8 {plus minus} 0.1 pH units. The purified enzyme is stimulated by Co{sup 3+} or Ni{sup 2+}. The enzyme utilizes 11-deoxycorticosterone, corticosterone, and 17-deoxycortisol as substrate but not cortisol, tetrahydrocortisol, and prednisolone. Tritium-water exchange of (21S)-(21-{sup 3}H)DOC is a pseudo-first-order reaction; 21-{sup 3}H exchange from the 21R isomer proceeds with first-order kinetics only after a lag associated with its epimerization to the 21S form.

  13. Secretion of protein disulphide isomerase AGR2 confers tumorigenic properties

    PubMed Central

    Fessart, Delphine; Domblides, Charlotte; Avril, Tony; Eriksson, Leif A; Begueret, Hugues; Pineau, Raphael; Malrieux, Camille; Dugot-Senant, Nathalie; Lucchesi, Carlo; Chevet, Eric; Delom, Frederic

    2016-01-01

    The extracellular matrix (ECM) plays an instrumental role in determining the spatial orientation of epithelial polarity and the formation of lumens in glandular tissues during morphogenesis. Here, we show that the Endoplasmic Reticulum (ER)-resident protein anterior gradient-2 (AGR2), a soluble protein-disulfide isomerase involved in ER protein folding and quality control, is secreted and interacts with the ECM. Extracellular AGR2 (eAGR2) is a microenvironmental regulator of epithelial tissue architecture, which plays a role in the preneoplastic phenotype and contributes to epithelial tumorigenicity. Indeed, eAGR2, is secreted as a functionally active protein independently of its thioredoxin-like domain (CXXS) and of its ER-retention domain (KTEL), and is sufficient, by itself, to promote the acquisition of invasive and metastatic features. Therefore, we conclude that eAGR2 plays an extracellular role independent of its ER function and we elucidate this gain-of-function as a novel and unexpected critical ECM microenvironmental pro-oncogenic regulator of epithelial morphogenesis and tumorigenesis. DOI: http://dx.doi.org/10.7554/eLife.13887.001 PMID:27240165

  14. Crystal Structure of Triosephosphate Isomerase from Trypanosoma cruzi in Hexane

    NASA Astrophysics Data System (ADS)

    Gao, Xiu-Gong; Maldonado, Ernesto; Perez-Montfort, Ruy; Garza-Ramos, Georgina; Tuena de Gomez-Puyou, Marietta; Gomez-Puyou, Armando; Rodriguez-Romero, Adela

    1999-08-01

    To gain insight into the mechanisms of enzyme catalysis in organic solvents, the x-ray structure of some monomeric enzymes in organic solvents was determined. However, it remained to be explored whether the structure of oligomeric proteins is also amenable to such analysis. The field acquired new perspectives when it was proposed that the x-ray structure of enzymes in nonaqueous media could reveal binding sites for organic solvents that in principle could represent the starting point for drug design. Here, a crystal of the dimeric enzyme triosephosphate isomerase from the pathogenic parasite Trypanosoma cruzi was soaked and diffracted in hexane and its structure solved at 2- angstrom resolution. Its overall structure and the dimer interface were not altered by hexane. However, there were differences in the orientation of the side chains of several amino acids, including that of the catalytic Glu-168 in one of the monomers. No hexane molecules were detected in the active site or in the dimer interface. However, three hexane molecules were identified on the surface of the protein at sites, which in the native crystal did not have water molecules. The number of water molecules in the hexane structure was higher than in the native crystal. Two hexanes localized at <4 angstrom from residues that form the dimer interface; they were in close proximity to a site that has been considered a potential target for drug design.

  15. Plant phosphomannose isomerase as a selectable marker for rice transformation.

    PubMed

    Hu, Lei; Li, Hao; Qin, Ruiying; Xu, Rongfang; Li, Juan; Li, Li; Wei, Pengcheng; Yang, Jianbo

    2016-01-01

    The E. coli phosphomannose isomerase (EcPMI) gene is widely used as a selectable marker gene (SMG) in mannose (Man) selection-based plant transformation. Although some plant species exhibit significant PMI activity and active PMIs were even identified in Man-sensitive plants, whether plant PMIs can be used as SMGs remains unclear. In this study, we isolated four novel PMI genes from Chlorella variabilis and Oryza sativa. Their isoenzymatic activities were examined in vitro and compared with that of EcPMI. The active plant PMIs were separately constructed into binary vectors as SMGs and then transformed into rice via Agrobacterium. In both Indica and Japonica subspecies, our results indicated that the plant PMIs could select and produce transgenic plants in a pattern similar to that of EcPMI. The transgenic plants exhibited an accumulation of plant PMI transcripts and enhancement of the in vivo PMI activity. Furthermore, a gene of interest was successfully transformed into rice using the plant PMIs as SMGs. Thus, novel SMGs for Man selection were isolated from plants, and our analysis suggested that PMIs encoding active enzymes might be common in plants and could potentially be used as appropriate genetic elements in cisgenesis engineering. PMID:27174847

  16. Catalytic mechanism of a retinoid isomerase essential for vertebrate vision

    SciTech Connect

    Kiser, Philip D.; Zhang, Jianye; Badiee, Mohsen; Li, Qingjiang; Shi, Wuxian; Sui, Xuewu; Golczak, Marcin; Tochtrop, Gregory P.; Palczewski, Krzysztof

    2015-04-20

    Visual function in vertebrates is dependent on the membrane-bound retinoid isomerase RPE65, an essential component of the retinoid cycle pathway that regenerates 11-cis-retinal for rod and cone opsins. The mechanism by which RPE65 catalyzes stereoselective retinoid isomerization has remained elusive because of uncertainty about how retinoids bind to its active site. Here we present crystal structures of RPE65 in complex with retinoid-mimetic compounds, one of which is in clinical trials for the treatment of age-related macular degeneration. The structures reveal the active site retinoid-binding cavity located near the membrane-interacting surface of the enzyme as well as an Fe-bound palmitate ligand positioned in an adjacent pocket. With the geometry of the RPE65–substrate complex clarified, we delineate a mechanism of catalysis that reconciles the extensive biochemical and structural research on this enzyme. These data provide molecular foundations for understanding a key process in vision and pharmacological inhibition of RPE65 with small molecules.

  17. Levels of Phosphate Esters in Spirodela

    PubMed Central

    Bieleski, R. L.

    1968-01-01

    The duckweed Spirodela oligorrhiza was grown in sterile nutrient solutions that contained 1 mm phosphate-32P at various specific activities. In solutions with activities higher than 2 μc per μmole per ml, plant growth was inhibited after a time, and the physical appearance of the plants was affected. The critical level of radiation, at which growth was first affected, corresponded to 5 kilorads. Plants were grown for 9 days (5 generations) in a culture solution containing phosphate at 0.5 μc per μmole per ml (radiation load approx 0.5 kilorads) so that all phosphorus-containing materials in the tissue became uniformly labeled. The various radioactive compounds were extracted, chromatographed, identified, and their radioactivity was measured. From this radioactivity plus the specific activity of the supplied phosphate, the amount of each compound was calculated. The data constitute a complete balance-sheet for phosphorus in a plant tissue. The identity of 98% of the phosphorus in the tissue was determined. Inorganic phosphate (32,700 mμmoles/g fr wt) was the predominant phosphorus-containing compound; RNA (5100 mμmoles P/g fr wt) was the main organic phosphate; phosphatidyl choline (1600 mμmoles/g fr wt) was the main phospholipid, and glucose-6-phosphate (500 mμmoles/g fr wt) the main acid-soluble phosphate ester. Amounts of other phosphorus compounds are given. Images PMID:16656910

  18. Co-expression of Triosephosphate Isomerase, Fructose-1, 6-bisphosphate Aldolase and Fructose-1, 6-bisphosphatase in E.coli.

    PubMed

    Tang, Gong-Li; Yang, Chun-Song; Bao, Jian-Shao; Wang, Yan-Fang; Chen, Hai-Bao; Shi, Ding-Ji; Liu, Feng-Long

    2001-01-01

    To establish a way to control or to decrease the daily increasing concentration of atmospheric CO(2), metabolically engineering Cyanobacteria was taken for the improvement of its efficiency of photosynthetic CO(2) fixation. As a preliminary stage of this study, three genes coding for three important Calvin cycle enzymes, i.e. triosephosphate isomerase (TPI), fructose-1, 6-bisphosphate aldolase(FBP aldolase),and fructose-1, 6-bisphosphatase(FBPase), respectively, have been cloned into one plasmid, pTrcFAT, which is controlled by promoter trc. Successful co-transcriptional expression of these three genes resulted inhigh yields of these enzymes under the induction of 0.25 mmol/L IPTG. Bioassay showed that the expressed enzymes from one liter of culture could directly catalyze DHAP conversion into 700 &mgr;mol of fructose-6-phosphate (F-6-P) per one minute. Furthermore, in order to introduce the three genes co-expression system into Cyanobacteria, a shuttle plasmid between E.coli and Cyanobacteria was constructed using plasmid pTrcFAT and a shuttle vector pDC-8, forming ashuttle plasmid pDCFAT-2 containing a dimer of the three genes co-expression operator. Successful co-expression in E.coli of pDCFAT-2 with higher full activity has been obtained. This shuttle was used to transform of Cyanobacteria Synechococcus sp. PCC 7942, and a few positive colonies were obtained. PMID:12053203

  19. Cell surface thiol isomerases may explain the platelet-selective action of S-nitrosoglutathione.

    PubMed

    Xiao, Fang; Gordge, Michael P

    2011-10-30

    S-nitrosoglutathione (GSNO) at low concentration inhibits platelet aggregation without causing vasodilation, suggesting platelet-selective nitric oxide delivery. The mechanism of this selectivity is unknown, but may involve cell surface thiol isomerases, in particular protein disulphide isomerase (csPDI) (EC 5.3.4.1). We have now compared csPDI expression and activity on platelets, endothelial cells and vascular smooth muscle cells, and the dependence on thiol reductase activity of these cell types for NO uptake from GSNO. csPDI expression was measured by flow cytometry and its reductase activity using the pseudosubstrate dieosin glutathione disulphide. This activity assay was adapted and validated for 96-well plate format. Flow cytometry revealed csPDI on all three cell types, but percentage positivity of expression was higher on platelets than on vascular cells. Consistent with this, thiol isomerase-related reductase activity was higher on platelets (P<0.01), and cellular activation (with either phorbol myristate acetate or ionomycin) increased csPDI activity on both platelets and smooth muscle cells, but not on endothelium. Intracellular NO delivery from GSNO was greater in platelets than in vascular cells (P<0.002), and was more sensitive to thiol isomerase inhibition using phenylarsine oxide (P<0.05). Increased surface thiol isomerase activity on platelets, compared with cells of the vascular wall, may explain the platelet-selective actions of GSNO and help define its antithrombotic potential. PMID:21642008

  20. Glucose metabolism and cardiac hypertrophy

    PubMed Central

    Kolwicz, Stephen C.; Tian, Rong

    2011-01-01

    The most notable change in the metabolic profile of hypertrophied hearts is an increased reliance on glucose with an overall reduced oxidative metabolism, i.e. a reappearance of the foetal metabolic pattern. In animal models, this change is attributed to the down-regulation of the transcriptional cascades promoting gene expression for fatty acid oxidation and mitochondrial oxidative phosphorylation in adult hearts. Impaired myocardial energetics in cardiac hypertrophy also triggers AMP-activated protein kinase (AMPK), leading to increased glucose uptake and glycolysis. Aside from increased reliance on glucose as an energy source, changes in other glucose metabolism pathways, e.g. the pentose phosphate pathway, the glucosamine biosynthesis pathway, and anaplerosis, are also noted in the hypertrophied hearts. Studies using transgenic mouse models and pharmacological compounds to mimic or counter the switch of substrate preference in cardiac hypertrophy have demonstrated that increased glucose metabolism in adult heart is not harmful and can be beneficial when it provides sufficient fuel for oxidative metabolism. However, improvement in the oxidative capacity and efficiency rather than the selection of the substrate is likely the ultimate goal for metabolic therapies. PMID:21502371

  1. Radiometric assays for glycerol, glucose, and glycogen.

    PubMed

    Bradley, D C; Kaslow, H R

    1989-07-01

    We have developed radiometric assays for small quantities of glycerol, glucose and glycogen, based on a technique described by Thorner and Paulus (1971, J. Biol. Chem. 246, 3885-3894) for the measurement of glycerokinase activity. In the glycerol assay, glycerol is phosphorylated with [32P]ATP and glycerokinase, residual [32P]ATP is hydrolyzed by heating in acid, and free [32P]phosphate is removed by precipitation with ammonium molybdate and triethylamine. Standard dose-response curves were linear from 50 to 3000 pmol glycerol with less than 3% SD in triplicate measurements. Of the substances tested for interference, only dihydroxyacetone gave a slight false positive signal at high concentration. When used to measure glycerol concentrations in serum and in media from incubated adipose tissue, the radiometric glycerol assay correlated well with a commonly used spectrophotometric assay. The radiometric glucose assay is similar to the glycerol assay, except that glucokinase is used instead of glycerokinase. Dose response was linear from 5 to 3000 pmol glucose with less than 3% SD in triplicate measurements. Glucosamine and N-acetylglucosamine gave false positive signals when equimolar to glucose. When glucose concentrations in serum were measured, the radiometric glucose assay agreed well with hexokinase/glucose-6-phosphate dehydrogenase (H/GDH)-based and glucose oxidase/H2O2-based glucose assays. The radiometric method for glycogen measurement incorporates previously described isolation and digestion techniques, followed by the radiometric assay of free glucose. When used to measure glycogen in mouse epididymal fat pads, the radiometric glycogen assay correlated well with the H/GDH-based glycogen assay. All three radiometric assays offer several practical advantages over spectral assays. PMID:2817333

  2. Radiometric assays for glycerol, glucose, and glycogen

    SciTech Connect

    Bradley, D.C.; Kaslow, H.R. )

    1989-07-01

    We have developed radiometric assays for small quantities of glycerol, glucose and glycogen, based on a technique described by Thorner and Paulus for the measurement of glycerokinase activity. In the glycerol assay, glycerol is phosphorylated with (32P)ATP and glycerokinase, residual (32P)ATP is hydrolyzed by heating in acid, and free (32P)phosphate is removed by precipitation with ammonium molybdate and triethylamine. Standard dose-response curves were linear from 50 to 3000 pmol glycerol with less than 3% SD in triplicate measurements. Of the substances tested for interference, only dihydroxyacetone gave a slight false positive signal at high concentration. When used to measure glycerol concentrations in serum and in media from incubated adipose tissue, the radiometric glycerol assay correlated well with a commonly used spectrophotometric assay. The radiometric glucose assay is similar to the glycerol assay, except that glucokinase is used instead of glycerokinase. Dose response was linear from 5 to 3000 pmol glucose with less than 3% SD in triplicate measurements. Glucosamine and N-acetylglucosamine gave false positive signals when equimolar to glucose. When glucose concentrations in serum were measured, the radiometric glucose assay agreed well with hexokinase/glucose-6-phosphate dehydrogenase (H/GDH)-based and glucose oxidase/H2O2-based glucose assays. The radiometric method for glycogen measurement incorporates previously described isolation and digestion techniques, followed by the radiometric assay of free glucose. When used to measure glycogen in mouse epididymal fat pads, the radiometric glycogen assay correlated well with the H/GDH-based glycogen assay. All three radiometric assays offer several practical advantages over spectral assays.

  3. MondoA Senses Non-glucose Sugars

    PubMed Central

    Stoltzman, Carrie A.; Kaadige, Mohan R.; Peterson, Christopher W.; Ayer, Donald E.

    2011-01-01

    Glucose is required for cell growth and proliferation. The MondoA·Mlx transcription factor is glucose-responsive and accumulates in the nucleus by sensing glucose 6-phosphate. One direct and glucose-induced target of MondoA·Mlx complexes is thioredoxin-interacting protein (TXNIP). TXNIP is a potent negative regulator of glucose uptake, and hence its regulation by MondoA·Mlx triggers a feedback loop that restricts glucose uptake. This feedback loop is similar to the “hexose transport curb” first described almost 30 years ago. We show here that MondoA responds to the non-glucose hexoses, allose, 3-O-methylglucose, and glucosamine by accumulating in the nucleus and activating TXNIP transcription. The metabolic inhibitor 3-bromopyruvate blocks the transcriptional response to allose and 3-O-methylglucose, indicating that their metabolism, or a parallel pathway, is required to stimulate MondoA activity. Our dissection of the hexosamine biosynthetic pathway suggests that in addition to sensing glucose 6-phosphate, MondoA can also sense glucosamine 6-phosphate. Analysis of glucose uptake in wild-type, MondoA-null, or TXNIP-null murine embryonic fibroblasts indicates a role for the MondoA-TXNIP regulatory circuit in the hexose transport curb, although other redundant pathways also contribute. PMID:21908621

  4. The SLC37 Family of Sugar-Phosphate/Phosphate Exchangers

    PubMed Central

    Chou, Janice Y.; Mansfield, Brian C.

    2014-01-01

    The SLC37 family members are endoplasmic reticulum (ER)-associated sugar-phosphate/phosphate (Pi) exchangers. Three of the four members, SLC37A1, SLC37A2, and SLC37A4, function as Pi-linked glucose-6-phosphate (G6P) antiporters catalyzing G6P:Pi and Pi:Pi exchanges. The activity of SLC37A3 is unknown. SLC37A4, better known as the G6P transporter (G6PT), has been extensively characterized, functionally and structurally, and is the best characterized family member. G6PT contains 10 transmembrane helices with both N and C termini facing the cytoplasm. The primary in vivo function of the G6PT protein is to translocate G6P from the cytoplasm into the ER lumen where it couples with either the liver/kidney/intestine-restricted glucose-6-phosphatase-α (G6Pase-α or G6PC) or the ubiquitously expressed G6Pase-β (or G6PC3) to hydrolyze G6P to glucose and Pi. The G6PT/G6Pase-α complex maintains interprandial glucose homeostasis, and the G6PT/G6Pase-β complex maintains neutro-phil energy homeostasis and functionality. G6PT is highly selective for G6P and is competitively inhibited by cholorogenic acid and its derivatives. Neither SLC37A1 nor SLC37A2 can couple functionally with G6Pase-α or G6Pase-β, and the antiporter activities of SLC37A1 or SLC37A2 are not inhibited by cholorogenic acid. Deficiencies in G6PT cause glycogen storage disease type Ib (GSD-Ib), a metabolic and immune disorder. To date, 91 separate SLC37A4 mutations, including 39 missense mutations, have been identified in GSD-Ib patients. Characterization of missense mutations has yielded valuable information on functionally important residues in the G6PT protein. The biological roles of the other SLC37 proteins remain to be determined and deficiencies have not yet been correlated to diseases. PMID:24745989

  5. Glucose Transporters in Cardiac Metabolism and Hypertrophy

    PubMed Central

    Shao, Dan; Tian, Rong

    2016-01-01

    The heart is adapted to utilize all classes of substrates to meet the high-energy demand, and it tightly regulates its substrate utilization in response to environmental changes. Although fatty acids are known as the predominant fuel for the adult heart at resting stage, the heart switches its substrate preference toward glucose during stress conditions such as ischemia and pathological hypertrophy. Notably, increasing evidence suggests that the loss of metabolic flexibility associated with increased reliance on glucose utilization contribute to the development of cardiac dysfunction. The changes in glucose metabolism in hypertrophied hearts include altered glucose transport and increased glycolysis. Despite the role of glucose as an energy source, changes in other nonenergy producing pathways related to glucose metabolism, such as hexosamine biosynthetic pathway and pentose phosphate pathway, are also observed in the diseased hearts. This article summarizes the current knowledge regarding the regulation of glucose transporter expression and translocation in the heart during physiological and pathological conditions. It also discusses the signaling mechanisms governing glucose uptake in cardiomyocytes, as well as the changes of cardiac glucose metabolism under disease conditions. PMID:26756635

  6. Glycogen contains phosphodiester groups that can be introduced by UDPglucose: glycogen glucose 1-phosphotransferase.

    PubMed

    Lomako, J; Lomako, W M; Whelan, W J; Marchase, R B

    1993-08-30

    Rabbit-muscle glycogen contains covalently bound phosphorus, equivalent to 1 phosphate group per 208 glucose residues. This often disputed, minor component was previously thought to represent a phosphomonoester group at C-6 of a glucose residue. Here we show that more than half the phosphorus is present as a phosphodiester, the remainder being monoester. A novel enzyme activity has been found in muscle that can account for the presence of the phosphodiester in glycogen. This is a UDPglucose: glycogen glucose 1-phosphotransferase that positions glucose 1-phosphate on C-6 of glucose residues in glycogen, forming a diester. The phosphomonoester groups present may arise by removal of the glucose residue originally transferred as glucose 1-phosphate. PMID:8396041

  7. Heterologous expression and characterization of bacterial 2-C-methyl-D-erythritol-4-phosphate pathway in Saccharomyces cerevisiae.

    PubMed

    Carlsen, Simon; Ajikumar, Parayil Kumaran; Formenti, Luca Riccardo; Zhou, Kang; Phon, Too Heng; Nielsen, Michael Lynge; Lantz, Anna Eliasson; Kielland-Brandt, Morten C; Stephanopoulos, Gregory

    2013-07-01

    Transfer of a biosynthetic pathway between evolutionary distant organisms can create a metabolic shunt capable of bypassing the native regulation of the host organism, hereby improving the production of secondary metabolite precursor molecules for important natural products. Here, we report the engineering of Escherichia coli genes encoding the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway into the genome of Saccharomyces cerevisiae and the characterization of intermediate metabolites synthesized by the MEP pathway in yeast. Our UPLC-MS analysis of the MEP pathway metabolites from engineered yeast showed that the pathway is active until the synthesis of 2-C-methyl-D-erythritol-2,4-cyclodiphosphate, but appears to lack functionality of the last two steps of the MEP pathway, catalyzed by the [4Fe-4S] iron sulfur cluster proteins encoded by ispG and ispH. In order to functionalize the last two steps of the MEP pathway, we co-expressed the genes for the E. coli iron sulfur cluster (ISC) assembly machinery. By deleting ERG13, thereby incapacitating the mevalonate pathway, in conjunction with labeling experiments with U-¹³C₆ glucose and growth experiments, we found that the ISC assembly machinery was unable to functionalize ispG and ispH. However, we have found that leuC and leuD, encoding the heterodimeric iron-sulfur cluster protein, isopropylmalate isomerase, can complement the S. cerevisiae leu1 auxotrophy. To our knowledge, this is the first time a bacterial iron-sulfur cluster protein has been functionally expressed in the cytosol of S. cerevisiae under aerobic conditions and shows that S. cerevisiae has the capability to functionally express at least some bacterial iron-sulfur cluster proteins in its cytosol. PMID:23636690

  8. Locating active-site hydrogen atoms in d-xylose isomerase: Time-of-flight neutron diffraction

    PubMed Central

    Katz, Amy K.; Li, Xinmin; Carrell, H. L.; Hanson, B. Leif; Langan, Paul; Coates, Leighton; Schoenborn, Benno P.; Glusker, Jenny P.; Bunick, Gerard J.

    2006-01-01

    Time-of-flight neutron diffraction has been used to locate hydrogen atoms that define the ionization states of amino acids in crystals of d-xylose isomerase. This enzyme, from Streptomyces rubiginosus, is one of the largest enzymes studied to date at high resolution (1.8 Å) by this method. We have determined the position and orientation of a metal ion-bound water molecule that is located in the active site of the enzyme; this water has been thought to be involved in the isomerization step in which d-xylose is converted to d-xylulose or d-glucose to d-fructose. It is shown to be water (rather than a hydroxyl group) under the conditions of measurement (pH 8.0). Our analyses also reveal that one lysine probably has an −NH2-terminal group (rather than NH3+). The ionization state of each histidine residue also was determined. High-resolution x-ray studies (at 0.94 Å) indicate disorder in some side chains when a truncated substrate is bound and suggest how some side chains might move during catalysis. This combination of time-of-flight neutron diffraction and x-ray diffraction can contribute greatly to the elucidation of enzyme mechanisms. PMID:16707576

  9. Glucose metabolism and hexosamine pathway regulate oncogene-induced senescence.

    PubMed

    Gitenay, D; Wiel, C; Lallet-Daher, H; Vindrieux, D; Aubert, S; Payen, L; Simonnet, H; Bernard, D

    2014-01-01

    Oncogenic stress-induced senescence (OIS) prevents the ability of oncogenic signals to induce tumorigenesis. It is now largely admitted that the mitogenic effect of oncogenes requires metabolic adaptations to respond to new energetic and bio constituent needs. Yet, whether glucose metabolism affects OIS response is largely unknown. This is largely because of the fact that most of the OIS cellular models are cultivated in glucose excess. In this study, we used human epithelial cells, cultivated without glucose excess, to study alteration and functional role of glucose metabolism during OIS. We report a slowdown of glucose uptake and metabolism during OIS. Increasing glucose metabolism by expressing hexokinase2 (HK2), which converts glucose to glucose-6-phosphate (G6P), favors escape from OIS. Inversely, expressing a glucose-6-phosphatase, [corrected] pharmacological inhibition of HK2, or adding nonmetabolizable glucose induced a premature senescence. Manipulations of various metabolites covering G6P downstream pathways (hexosamine, glycolysis, and pentose phosphate pathways) suggest an unexpected role of the hexosamine pathway in controlling OIS. Altogether, our results show that decreased glucose metabolism occurs during and participates to OIS. PMID:24577087

  10. Identification of fibrillogenic regions in human triosephosphate isomerase

    PubMed Central

    Carcamo-Noriega, Edson N.

    2016-01-01

    Background. Amyloid secondary structure relies on the intermolecular assembly of polypeptide chains through main-chain interaction. According to this, all proteins have the potential to form amyloid structure, nevertheless, in nature only few proteins aggregate into toxic or functional amyloids. Structural characteristics differ greatly among amyloid proteins reported, so it has been difficult to link the fibrillogenic propensity with structural topology. However, there are ubiquitous topologies not represented in the amyloidome that could be considered as amyloid-resistant attributable to structural features, such is the case of TIM barrel topology. Methods. This work was aimed to study the fibrillogenic propensity of human triosephosphate isomerase (HsTPI) as a model of TIM barrels. In order to do so, aggregation of HsTPI was evaluated under native-like and destabilizing conditions. Fibrillogenic regions were identified by bioinformatics approaches, protein fragmentation and peptide aggregation. Results. We identified four fibrillogenic regions in the HsTPI corresponding to the β3, β6, β7 y α8 of the TIM barrel. From these, the β3-strand region (residues 59–66) was highly fibrillogenic. In aggregation assays, HsTPI under native-like conditions led to amorphous assemblies while under partially denaturing conditions (urea 3.2 M) formed more structured aggregates. This slightly structured aggregates exhibited residual cross-β structure, as demonstrated by the recognition of the WO1 antibody and ATR-FTIR analysis. Discussion. Despite the fibrillogenic regions present in HsTPI, the enzyme maintained under native-favoring conditions displayed low fibrillogenic propensity. This amyloid-resistance can be attributed to the three-dimensional arrangement of the protein, where β-strands, susceptible to aggregation, are protected in the core of the molecule. Destabilization of the protein structure may expose inner regions promoting β-aggregation, as well as the

  11. Purification, characterization and catalytic properties of human sterol 8-isomerase.

    PubMed Central

    Nes, W David; Zhou, Wenxu; Dennis, Allen L; Li, Haoxia; Jia, Zhonghua; Keith, Richard A; Piser, Timothy M; Furlong, Stephen T

    2002-01-01

    CHO 2, encoding human sterol 8-isomerase (hSI), was introduced into plasmids pYX213 or pET23a. The resulting native protein was overexpressed in erg 2 yeast cells and purified to apparent homogeneity. The enzyme exhibited a K (m) of 50 microM and a turnover number of 0.423 s(-1) for zymosterol, an isoelectric point of 7.70, a native molecular mass of 107000 Da and was tetrameric. The structural features of zymosterol provided optimal substrate acceptability. Biomimetic studies of acid-catalysed isomerization of zymosterol resulted in formation of cholest-8(14)-enol, whereas the enzyme-generated product was a Delta(7)-sterol, suggesting absolute stereochemical control of the reaction by hSI. Using (2)H(2)O and either zymosterol or cholesta-7,24-dienol as substrates, the reversibility of the reaction was confirmed by GC-MS of the deuterated products. The positional specific incorporation of deuterium at C-9alpha was established by a combination of (1)H- and (13)C-NMR analyses of the enzyme-generated cholesta-7,24-dienol. Kinetic analyses indicated the reaction equilibrium ( K (eq)=14; DeltaG(o')=-6.5 kJ/mol) for double-bond isomerization favoured the forward direction, Delta(8) to Delta(7). Treatment of hSI with different high-energy intermediate analogues produced the following dissociation constants ( K (i)): emopamil (2 microM)=tamoxifen (1 microM)=tridemorph (1 microM)<25-azacholesterol (21 microM)

  12. Identification of fibrillogenic regions in human triosephosphate isomerase.

    PubMed

    Carcamo-Noriega, Edson N; Saab-Rincon, Gloria

    2016-01-01

    Background. Amyloid secondary structure relies on the intermolecular assembly of polypeptide chains through main-chain interaction. According to this, all proteins have the potential to form amyloid structure, nevertheless, in nature only few proteins aggregate into toxic or functional amyloids. Structural characteristics differ greatly among amyloid proteins reported, so it has been difficult to link the fibrillogenic propensity with structural topology. However, there are ubiquitous topologies not represented in the amyloidome that could be considered as amyloid-resistant attributable to structural features, such is the case of TIM barrel topology. Methods. This work was aimed to study the fibrillogenic propensity of human triosephosphate isomerase (HsTPI) as a model of TIM barrels. In order to do so, aggregation of HsTPI was evaluated under native-like and destabilizing conditions. Fibrillogenic regions were identified by bioinformatics approaches, protein fragmentation and peptide aggregation. Results. We identified four fibrillogenic regions in the HsTPI corresponding to the β3, β6, β7 y α8 of the TIM barrel. From these, the β3-strand region (residues 59-66) was highly fibrillogenic. In aggregation assays, HsTPI under native-like conditions led to amorphous assemblies while under partially denaturing conditions (urea 3.2 M) formed more structured aggregates. This slightly structured aggregates exhibited residual cross-β structure, as demonstrated by the recognition of the WO1 antibody and ATR-FTIR analysis. Discussion. Despite the fibrillogenic regions present in HsTPI, the enzyme maintained under native-favoring conditions displayed low fibrillogenic propensity. This amyloid-resistance can be attributed to the three-dimensional arrangement of the protein, where β-strands, susceptible to aggregation, are protected in the core of the molecule. Destabilization of the protein structure may expose inner regions promoting β-aggregation, as well as the

  13. Purification, characterization, and amino acid sequencing of a. delta. /sup 5/-3-oxosteroid isomerase from Pseudomonas putida biotype B

    SciTech Connect

    Linden, K.G.

    1986-01-01

    Studies were performed on the ..delta../sup 5/-3-oxosteroid isomerase from Pseudomonas putida biotype B. The studies have involved three broad areas: improvement in the purification of the enzyme, further characterization of the purified enzyme, and completion of the amino acid sequence of the enzyme. For the purification of the enzyme, techniques for removing the isomerase from whole cells were studied, the effects of ionic strength on the binding of the isomerase to steroidal affinity resins was explored, and a new affinity resin was developed. Absorption spectra and the proton NMR spectra of the isomerase were obtained. Amino acid sequencing of the oxosteroid isomerase indicates that the enzyme is a dimeric protein consisting of two identical subunits each consisting of a polypeptide chain of 131 residues and a M/sub r/ = 14,536.

  14. Characterization of the microsomal steroid-8-ene isomerase of cholesterol biosynthesis.

    PubMed

    Yamaga, N; Gaylor, J L

    1978-03-01

    Rat liver microsomes contain an enzyme that catalyzes the isomerization of the nuclear double bond of steroids from the 8(9) position to the 7(8) position. The enzyme is most active with zymosterol, 5alpha-cholesta-8,24-dien-3beta-ol, which is a precursor of cholesterol. Properties of the microsomal isomerase have now been studied, and preliminary data are reported on both regulation of enzymic activity and first steps in the solubilization of the enzyme from membranes. After a brief lag period, the velocity of isomerase is relatively constant for about 5 min of incubation, and then isomerization subsides. The apparent Michaelis constant (52-70 micro M) is difficult to determine accurately, due to these complex kinetic changes. V(max) is 4.0-4.7 nmol/min per mg of microsomal protein. The apparent specific activity is more than ten times that of liver microsomal methyl sterol oxidase. The maximal specific activity of microsomal isomerase is approximately doubled when rats are fed an intestinal bile acid sequestrant, cholestyramine. Changes in specific activity of isomerase parallel changes in activities of other microsomal enzymes of cholesterol biosynthesis, such as 3-hydroxy-3-methylglutaryl-CoA reductase and 4-methyl sterol oxidase. Isomerase activity is destroyed by phospholipase A digestion, high concentration of bile salts, and solvent extraction, all of which are known either to remove phospholipid or to alter microsomal membrane integrity. On the other hand, isomerase remains active in the presence of a mild, nonionic detergent, Triton WR-1339; thus, solubilization with nonionic detergents is under study. PMID:650094

  15. Expression of the Escherichia coli xylose isomerase gene in Saccharomyces cerevisiae

    SciTech Connect

    Sarthy, A.V.; McConaughy, B.L.; Lobo, Z.; Sundstrom, J.A.; Furlong, C.E.; Hall, B.D.

    1987-09-01

    Transformation of Saccharomyces cerevisiae by yeast expression plasmids bearing the Escherichia coli xylose isomerase gene leads to production of the protein. Western blotting experiments show that immunoreactive protein chains which comigrate with the E. coli enzyme are made in the transformant strains and that the amount produced parallels the copy number of the plasmid. When comparable amounts of immunologically cross-reactive xylose isomerase protein made in E. coli or S. cerevisiae were assayed for enzymatic activity, however, the yeast protein was at least 10/sup 3/-fold less active.

  16. Microbial solubilization of phosphate

    DOEpatents

    Rogers, R.D.; Wolfram, J.H.

    1993-10-26

    A process is provided for solubilizing phosphate from phosphate containing ore by treatment with microorganisms which comprises forming an aqueous mixture of phosphate ore, microorganisms operable for solubilizing phosphate from the phosphate ore and maintaining the aqueous mixture for a period of time and under conditions operable to effect the microbial solubilization process. An aqueous solution containing soluble phosphorus can be separated from the reacted mixture by precipitation, solvent extraction, selective membrane, exchange resin or gravity methods to recover phosphate from the aqueous solution. 6 figures.

  17. Microbial solubilization of phosphate

    DOEpatents

    Rogers, Robert D.; Wolfram, James H.

    1993-01-01

    A process is provided for solubilizing phosphate from phosphate containing ore by treatment with microorganisms which comprises forming an aqueous mixture of phosphate ore, microorganisms operable for solubilizing phosphate from the phosphate ore and maintaining the aqueous mixture for a period of time and under conditions operable to effect the microbial solubilization process. An aqueous solution containing soluble phosphorous can be separated from the reacted mixture by precipitation, solvent extraction, selective membrane, exchange resin or gravity methods to recover phosphate from the aqueous solution.

  18. The pentose phosphate pathway and cancer

    PubMed Central

    Patra, Krushna C.; Hay, Nissim

    2015-01-01

    The pentose phosphate pathway (PPP), which branches from glycolysis at the first committed step of glucose metabolism, is required for the synthesis of ribonucleotides and is a major source of NADPH. NADPH is required for and consumed during fatty acid synthesis and the scavenging of reactive oxygen species. Therefore, the PPP plays a pivotal role in helping glycolytic cancer cells to meet their anabolic demands and combat oxidative stress. Recently, several neoplastic lesions were shown to have evolved to facilitate the flux of glucose into the pentose phosphate pathway. This review summarizes the fundamental functions of the PPP, its regulation in cancer cells, and its importance in cancer cell metabolism and survival. PMID:25037503

  19. 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.

  20. Vanadate influence on metabolism of sugar phosphates in fungus Phycomyces blakesleeanus.

    PubMed

    Žižić, Milan; Živić, Miroslav; Maksimović, Vuk; Stanić, Marina; Križak, Strahinja; Antić, Tijana Cvetić; Zakrzewska, Joanna

    2014-01-01

    The biological and chemical basis of vanadium action in fungi is relatively poorly understood. In the present study, we investigate the influence of vanadate (V5+) on phosphate metabolism of Phycomyces blakesleeanus. Addition of V5+ caused increase of sugar phosphates signal intensities in 31P NMR spectra in vivo. HPLC analysis of mycelial phosphate extracts demonstrated increased concentrations of glucose 6 phosphate, fructose 6 phosphate, fructose 1, 6 phosphate and glucose 1 phosphate after V5+ treatment. Influence of V5+ on the levels of fructose 2, 6 phosphate, glucosamine 6 phosphate and glucose 1, 6 phosphate (HPLC), and polyphosphates, UDPG and ATP (31P NMR) was also established. I