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Sample records for starch phosphorylase mutational

  1. Mutation of the Plastidial α-Glucan Phosphorylase Gene in Rice Affects the Synthesis and Structure of Starch in the Endosperm[W

    PubMed Central

    Satoh, Hikaru; Shibahara, Kensuke; Tokunaga, Takashi; Nishi, Aiko; Tasaki, Mikako; Hwang, Seon-Kap; Okita, Thomas W.; Kaneko, Nanae; Fujita, Naoko; Yoshida, Mayumi; Hosaka, Yuko; Sato, Aya; Utsumi, Yoshinori; Ohdan, Takashi; Nakamura, Yasunori

    2008-01-01

    Plastidial phosphorylase (Pho1) accounts for ∼96% of the total phosphorylase activity in developing rice (Oryza sativa) seeds. From mutant stocks induced by N-methyl-N-nitrosourea treatment, we identified plants with mutations in the Pho1 gene that are deficient in Pho1. Strikingly, the size of mature seeds and the starch content in these mutants showed considerable variation, ranging from shrunken to pseudonormal. The loss of Pho1 caused smaller starch granules to accumulate and modified the amylopectin structure. Variation in the morphological and biochemical phenotype of individual seeds was common to all 15 pho1-independent homozygous mutant lines studied, indicating that this phenotype was caused solely by the genetic defect. The phenotype of the pho1 mutation was temperature dependent. While the mutant plants grown at 30°C produced mainly plump seeds at maturity, most of the seeds from plants grown at 20°C were shrunken, with a significant proportion showing severe reduction in starch accumulation. These results strongly suggest that Pho1 plays a crucial role in starch biosynthesis in rice endosperm at low temperatures and that one or more other factors can complement the function of Pho1 at high temperatures. PMID:18621947

  2. High phosphorylase activity is correlated with increased potato minituber formation and starch content during extended clinorotation

    NASA Astrophysics Data System (ADS)

    Nedukha, O. M.; Schnyukova, E. I.; Leach, J. E.

    2003-05-01

    The major purpose of these experiments were to investigate growth of potato storage organs and starch synthesis in minitubers at slow horizontal clinorotation (2 rpm), which partly mimics microgravity, and a secondary goal was to study the activity and localization of phosphorylase (EC 2.4.1.1) in storage parenchyma under these conditions. Miniplants of Solanum tuberosum L. (cv Adreta) were grown in culture for 30 days for both the vertical control and the horizontal clinorotation. During long-term clinorotation, an acceleration of minituber formation, and an increase of amyloplast number and size in storage parenchyma cells, as well as increased starch content, was observed in the minitubers. The differences among cytochemical reaction intensity, activity of phosphorylase, and carbohydrate content in storage parenchyma cells of minitubers grown in a horizontal clinostat were established by electron-cytochemical and biochemical methods. It is shown that high phosphorylase activity is correlated with increased starch content during extended clinorotation. The results demonstrate the increase in carbohydrate metabolism and possible accelerated growth of storage organs under the influence of microgravity, as mimicked by clinorotation; therefore, clinorotation can be used as a basis for future studies on mechanisms of starch synthesis under microgravity.

  3. Identification of the maize amyloplast stromal 112-kD protein as a plastidic starch phosphorylase.

    PubMed

    Yu, Y; Mu, H H; Wasserman, B P; Carman, G M

    2001-01-01

    Amyloplast is the site of starch synthesis in the storage tissue of maize (Zea mays). The amyloplast stroma contains an enriched group of proteins when compared with the whole endosperm. Proteins with molecular masses of 76 and 85 kD have been identified as starch synthase I and starch branching enzyme IIb, respectively. A 112-kD protein was isolated from the stromal fraction by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to tryptic digestion and amino acid sequence analysis. Three peptide sequences showed high identity to plastidic forms of starch phosphorylase (SP) from sweet potato, potato, and spinach. SP activity was identified in the amyloplast stromal fraction and was enriched 4-fold when compared with the activity in the whole endosperm fraction. Native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses showed that SP activity was associated with the amyloplast stromal 112-kD protein. In addition, antibodies raised against the potato plastidic SP recognized the amyloplast stromal 112-kD protein. The amyloplast stromal 112-kD SP was expressed in whole endosperm isolated from maize harvested 9 to 24 d after pollination. Results of affinity electrophoresis and enzyme kinetic analyses showed that the amyloplast stromal 112-kD SP preferred amylopectin over glycogen as a substrate in the synthetic reaction. The maize shrunken-4 mutant had reduced SP activity due to a decrease of the amyloplast stromal 112-kD enzyme.

  4. Overexpression of the Starch Phosphorylase-Like Gene (PHO3) in Lotus japonicus has a Profound Effect on the Growth of Plants and Reduction of Transitory Starch Accumulation

    PubMed Central

    Qin, Shanshan; Tang, Yuehui; Chen, Yaping; Wu, Pingzhi; Li, Meiru; Wu, Guojiang; Jiang, Huawu

    2016-01-01

    Two isoforms of starch phosphorylase (PHO; EC 2.4.1.1), plastidic PHO1 and cytosolic PHO2, have been found in all plants studied to date. Another starch phosphorylase-like gene, PHO3, which is an ortholog of Chlamydomonas PHOB, has been detected in some plant lineages. In this study, we identified three PHO isoform (LjPHO) genes in the Lotus japonicus genome. Expression of the LjPHO3 gene was observed in all tissues tested in L. japonicus, and the LjPHO3 protein was located in the chloroplast. Overexpression of LjPHO3 in L. japonicus resulted in a drastic decline in starch granule sizes and starch content in leaves. The LjPHO3 overexpression transgenic seedlings were smaller, and showed decreased pollen fertility and seed set rate. Our results suggest that LjPHO3 may participate in transitory starch metabolism in L. japonicus leaves, but its catalytic properties remain to be studied.

  5. Overexpression of the Starch Phosphorylase-Like Gene (PHO3) in Lotus japonicus has a Profound Effect on the Growth of Plants and Reduction of Transitory Starch Accumulation.

    PubMed

    Qin, Shanshan; Tang, Yuehui; Chen, Yaping; Wu, Pingzhi; Li, Meiru; Wu, Guojiang; Jiang, Huawu

    2016-01-01

    Two isoforms of starch phosphorylase (PHO; EC 2.4.1.1), plastidic PHO1 and cytosolic PHO2, have been found in all plants studied to date. Another starch phosphorylase-like gene, PHO3, which is an ortholog of Chlamydomonas PHOB, has been detected in some plant lineages. In this study, we identified three PHO isoform (LjPHO) genes in the Lotus japonicus genome. Expression of the LjPHO3 gene was observed in all tissues tested in L. japonicus, and the LjPHO3 protein was located in the chloroplast. Overexpression of LjPHO3 in L. japonicus resulted in a drastic decline in starch granule sizes and starch content in leaves. The LjPHO3 overexpression transgenic seedlings were smaller, and showed decreased pollen fertility and seed set rate. Our results suggest that LjPHO3 may participate in transitory starch metabolism in L. japonicus leaves, but its catalytic properties remain to be studied. PMID:27630651

  6. Overexpression of the Starch Phosphorylase-Like Gene (PHO3) in Lotus japonicus has a Profound Effect on the Growth of Plants and Reduction of Transitory Starch Accumulation

    PubMed Central

    Qin, Shanshan; Tang, Yuehui; Chen, Yaping; Wu, Pingzhi; Li, Meiru; Wu, Guojiang; Jiang, Huawu

    2016-01-01

    Two isoforms of starch phosphorylase (PHO; EC 2.4.1.1), plastidic PHO1 and cytosolic PHO2, have been found in all plants studied to date. Another starch phosphorylase-like gene, PHO3, which is an ortholog of Chlamydomonas PHOB, has been detected in some plant lineages. In this study, we identified three PHO isoform (LjPHO) genes in the Lotus japonicus genome. Expression of the LjPHO3 gene was observed in all tissues tested in L. japonicus, and the LjPHO3 protein was located in the chloroplast. Overexpression of LjPHO3 in L. japonicus resulted in a drastic decline in starch granule sizes and starch content in leaves. The LjPHO3 overexpression transgenic seedlings were smaller, and showed decreased pollen fertility and seed set rate. Our results suggest that LjPHO3 may participate in transitory starch metabolism in L. japonicus leaves, but its catalytic properties remain to be studied. PMID:27630651

  7. Identification of the Maize Amyloplast Stromal 112-kD Protein as a Plastidic Starch Phosphorylase12

    PubMed Central

    Yu, Ying; Mu, Helen He; Wasserman, Bruce P.; Carman, George M.

    2001-01-01

    Amyloplast is the site of starch synthesis in the storage tissue of maize (Zea mays). The amyloplast stroma contains an enriched group of proteins when compared with the whole endosperm. Proteins with molecular masses of 76 and 85 kD have been identified as starch synthase I and starch branching enzyme IIb, respectively. A 112-kD protein was isolated from the stromal fraction by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to tryptic digestion and amino acid sequence analysis. Three peptide sequences showed high identity to plastidic forms of starch phosphorylase (SP) from sweet potato, potato, and spinach. SP activity was identified in the amyloplast stromal fraction and was enriched 4-fold when compared with the activity in the whole endosperm fraction. Native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses showed that SP activity was associated with the amyloplast stromal 112-kD protein. In addition, antibodies raised against the potato plastidic SP recognized the amyloplast stromal 112-kD protein. The amyloplast stromal 112-kD SP was expressed in whole endosperm isolated from maize harvested 9 to 24 d after pollination. Results of affinity electrophoresis and enzyme kinetic analyses showed that the amyloplast stromal 112-kD SP preferred amylopectin over glycogen as a substrate in the synthetic reaction. The maize shrunken-4 mutant had reduced SP activity due to a decrease of the amyloplast stromal 112-kD enzyme. PMID:11154342

  8. Rice Endosperm Starch Phosphorylase (Pho1) Assembles with Disproportionating Enzyme (Dpe1) to Form a Protein Complex That Enhances Synthesis of Malto-oligosaccharides.

    PubMed

    Hwang, Seon-Kap; Koper, Kaan; Satoh, Hikaru; Okita, Thomas W

    2016-09-16

    Starch synthesis in cereal grain endosperm is dependent on the concerted actions of many enzymes. The starch plastidial phosphorylase (Pho1) plays an important role in the initiation of starch synthesis and in the maturation of starch granule in developing rice seeds. Prior evidence has suggested that the rice enzyme, OsPho1, may have a physical/functional interaction with other starch biosynthetic enzymes. Pulldown experiments showed that OsPho1 as well as OsPho1 devoid of its L80 region, a peptide unique to higher plant phosphorylases, captures disproportionating enzyme (OsDpe1). Interaction of the latter enzyme form with OsDpe1 indicates that the putative regulatory L80 is not responsible for multienzyme assembly. This heterotypic enzyme complex, determined at a molar ratio of 1:1, was validated by reciprocal co-immunoprecipitation studies of native seed proteins and by co-elution chromatographic and co-migration electrophoretic patterns of these enzymes in rice seed extracts. The OsPho1-OsDpe1 complex utilized a broader range of substrates for enhanced synthesis of larger maltooligosaccharides than each individual enzyme and significantly elevated the substrate affinities of OsPho1 at 30 °C. Moreover, the assembly with OsDpe1 enables OsPho1 to utilize products of transglycosylation reactions involving G1 and G3, sugars that it cannot catalyze directly. PMID:27502283

  9. Reduction of the plastidial phosphorylase in potato (Solanum tuberosum L.) reveals impact on storage starch structure during growth at low temperature.

    PubMed

    Orawetz, Tom; Malinova, Irina; Orzechowski, Slawomir; Fettke, Joerg

    2016-03-01

    Tubers of potato (Solanum tuberosum L.), one of the most important crops, are a prominent example for an efficient production of storage starch. Nevertheless, the synthesis of this storage starch is not completely understood. The plastidial phosphorylase (Pho1; EC 2.4.1.1) catalyzes the reversible transfer of glucosyl residues from glucose-1-phosphate to the non-reducing end of α-glucans with the release of orthophosphate. Thus, the enzyme is in principle able to act during starch synthesis. However, so far under normal growth conditions no alterations in tuber starch metabolism were observed. Based on analyses of other species and also from in vitro experiments with potato tuber slices it was supposed, that Pho1 has a stronger impact on starch metabolism, when plants grow under low temperature conditions. Therefore, we analyzed the starch content, granule size, as well as the internal structure of starch granules isolated from potato plants grown under low temperatures. Besides wild type, transgenic potato plants with a strong reduction in the Pho1 activity were analyzed. No significant alterations in starch content and granule size were detected. In contrast, when plants were cultivated at low temperatures the chain length distributions of the starch granules were altered. Thus, the granules contained more short glucan chains. That was not observed in the transgenic plants, revealing that Pho1 in wild type is involved in the formation of the short glucan chains, at least at low temperatures. PMID:26828405

  10. Reduction of the plastidial phosphorylase in potato (Solanum tuberosum L.) reveals impact on storage starch structure during growth at low temperature.

    PubMed

    Orawetz, Tom; Malinova, Irina; Orzechowski, Slawomir; Fettke, Joerg

    2016-03-01

    Tubers of potato (Solanum tuberosum L.), one of the most important crops, are a prominent example for an efficient production of storage starch. Nevertheless, the synthesis of this storage starch is not completely understood. The plastidial phosphorylase (Pho1; EC 2.4.1.1) catalyzes the reversible transfer of glucosyl residues from glucose-1-phosphate to the non-reducing end of α-glucans with the release of orthophosphate. Thus, the enzyme is in principle able to act during starch synthesis. However, so far under normal growth conditions no alterations in tuber starch metabolism were observed. Based on analyses of other species and also from in vitro experiments with potato tuber slices it was supposed, that Pho1 has a stronger impact on starch metabolism, when plants grow under low temperature conditions. Therefore, we analyzed the starch content, granule size, as well as the internal structure of starch granules isolated from potato plants grown under low temperatures. Besides wild type, transgenic potato plants with a strong reduction in the Pho1 activity were analyzed. No significant alterations in starch content and granule size were detected. In contrast, when plants were cultivated at low temperatures the chain length distributions of the starch granules were altered. Thus, the granules contained more short glucan chains. That was not observed in the transgenic plants, revealing that Pho1 in wild type is involved in the formation of the short glucan chains, at least at low temperatures.

  11. A mutation in polynucleotide phosphorylase from Escherichia coli impairing RNA binding and degradosome stability

    PubMed Central

    Regonesi, Maria Elena; Briani, Federica; Ghetta, Andrea; Zangrossi, Sandro; Ghisotti, Daniela; Tortora, Paolo; Dehò, Gianni

    2004-01-01

    Polynucleotide phosphorylase (PNPase), a 3′ to 5′ exonuclease encoded by pnp, plays a key role in Escherichia coli RNA decay. The enzyme, made of three identical 711 amino acid subunits, may also be assembled in the RNA degradosome, a heteromultimeric complex involved in RNA degradation. PNPase autogenously regulates its expression by promoting the decay of pnp mRNA, supposedly by binding at the 5′-untranslated leader region of an RNase III-processed form of this transcript. The KH and S1 RNA-binding domains at the C-terminus of the protein (amino acids 552–711) are thought to be involved in pnp mRNA recognition. Here we show that a G454D substitution in E.coli PNPase impairs autogenous regulation whereas it does not affect the catalytic activities of the enzyme. Although the mutation maps outside of the KH and S1 RNA-binding domains, analysis of the mutant protein revealed a defective RNA binding, thus suggesting that other determinants may be involved in PNPase–RNA interactions. The mutation also caused a looser association with the degradosome and an abnormal electrophoretic mobility in native gels. The latter feature suggests an altered structural conformation of PNPase, which may account for the properties of the mutant protein. PMID:14963263

  12. Site-Selective Ribosylation of Fluorescent Nucleobase Analogs Using Purine-Nucleoside Phosphorylase as a Catalyst: Effects of Point Mutations.

    PubMed

    Stachelska-Wierzchowska, Alicja; Wierzchowski, Jacek; Bzowska, Agnieszka; Wielgus-Kutrowska, Beata

    2015-12-28

    Enzymatic ribosylation of fluorescent 8-azapurine derivatives, like 8-azaguanine and 2,6-diamino-8-azapurine, with purine-nucleoside phosphorylase (PNP) as a catalyst, leads to N9, N8, and N7-ribosides. The final proportion of the products may be modulated by point mutations in the enzyme active site. As an example, ribosylation of the latter substrate by wild-type calf PNP gives N7- and N8-ribosides, while the N243D mutant directs the ribosyl substitution at N9- and N7-positions. The same mutant allows synthesis of the fluorescent N7-β-d-ribosyl-8-azaguanine. The mutated form of the E. coli PNP, D204N, can be utilized to obtain non-typical ribosides of 8-azaadenine and 2,6-diamino-8-azapurine as well. The N7- and N8-ribosides of the 8-azapurines can be analytically useful, as illustrated by N7-β-d-ribosyl-2,6-diamino-8-azapurine, which is a good fluorogenic substrate for mammalian forms of PNP, including human blood PNP, while the N8-riboside is selective to the E. coli enzyme.

  13. Induced mutations in the starch branching enzyme II (SBEII) genes increase amylose and resistant starch content in durum wheat

    PubMed Central

    Hazard, Brittany; Zhang, Xiaoqin; Colasuonno, Pasqualina; Uauy, Cristobal; Beckles, Diane M.; Dubcovsky, Jorge

    2016-01-01

    Starch is the largest component of the wheat (Triticum aestivum L.) grain and consists of approximately 70-80% amylopectin and 20-30% amylose. Amylopectin is a highly-branched, readily digested polysaccharide, whereas amylose has few branches and forms complexes that resist digestion and mimic dietary fiber (resistant starch). Down-regulation of the starch branching enzyme II (SBEII) gene by RNA interference (RNAi) was previously shown to increase amylose content in both hexaploid and tetraploid wheat. We generated ethyl methane sulphonate (EMS) mutants for the SBEIIa-A and SBEIIa-B homoeologs in the tetraploid durum wheat variety Kronos (T. turgidum ssp. durum L.). Single-gene mutants showed non-significant increases in amylose and resistant starch content, but a double mutant combining a SBEIIa-A knock-out mutation with a SBEIIa-B splice-site mutation showed a 22% increase in amylose content (P<0.0001) and a 115% increase in resistant starch content (P<0.0001). In addition, we obtained mutants for the A and B genome copies of the paralogous SBEIIb gene, mapped them 1-2 cM from SBEIIa, and generated double SBEIIa-SBEIIb mutants to study the effect of the SBEIIb gene in the absence of SBEIIa. These mutants are available to those interested in increasing amylose content and resistant starch in durum wheat. PMID:26924849

  14. Physiological aggregation of maltodextrin phosphorylase from Pyrococcus furiosus and its application in a process of batch starch degradation to alpha-D-glucose-1-phosphate.

    PubMed

    Nahálka, Jozef

    2008-04-01

    Maltodextrin phosphorylase from Pyrococcus furiosus (PF1535) was fused with the cellulose-binding domain of Clostridium cellulovorans serving as an aggregation module. After molecular cloning of the corresponding gene fusion construct and controlled expression in Escherichia coli BL21, 83% of total maltodextrin phosphorylase activity (0.24 U/mg of dry cell weight) was displayed in active inclusion bodies. These active inclusion bodies were easily isolated by nonionic detergent treatment and directly used for maltodextrin conversion to alpha-D-glucose-1-phosphate in a repetitive batch mode. Only 10% of enzyme activity was lost after ten conversion cycles at optimum conditions.

  15. Fatal infantile cardiac glycogenosis with phosphorylase kinase deficiency and a mutation in the gamma2-subunit of AMP-activated protein kinase.

    PubMed

    Akman, Hasan O; Sampayo, James N; Ross, Fiona A; Scott, John W; Wilson, Gregory; Benson, Lee; Bruno, Claudio; Shanske, Sara; Hardie, D Grahame; Dimauro, Salvatore

    2007-10-01

    A 10-wk-old infant girl with severe hypertrophy of the septal and atrial walls by cardiac ultrasound, developed progressive ventricular wall thickening and died of aspiration pneumonia at 5 mo of age. Postmortem examination revealed ventricular hypertrophy and massive atrial wall thickening due to glycogen accumulation. A skeletal muscle biopsy showed increased free glycogen and decreased activity of phosphorylase b kinase (PHK). The report of a pathogenic mutation (R531Q) in the gene (PRKAG2) encoding the gamma2 subunit of AMP-activated protein kinase (AMPK) in three infants with congenital hypertrophic cardiomyopathy, glycogen storage, and "pseudo PHK deficiency" prompted us to screen this gene in our patient. We found a novel (R384T) heterozygous mutation in PRKAG2, affecting an arginine residue in the N-terminal AMP-binding domain. Like R531Q, this mutation reduces the binding of AMP and ATP to the isolated nucleotide-binding domains, and prevents activation of the heterotrimer by metabolic stress in intact cells. The mutation was not found in DNA from the patient's father, the only available parent, and is likely to have arisen de novo. Our studies confirm that mutations in PRKAG2 can cause fatal infantile cardiomyopathy, often associated with apparent PHK deficiency.

  16. Use of advanced recombinant lines to study the impact and potential of mutations affecting starch synthesis in barley☆

    PubMed Central

    Howard, Thomas P.; Fahy, Brendan; Leigh, Fiona; Howell, Phil; Powell, Wayne; Greenland, Andy; Trafford, Kay; Smith, Alison M.

    2014-01-01

    The effects on barley starch and grain properties of four starch synthesis mutations were studied during the introgression of the mutations from diverse backgrounds into an elite variety. The lys5f (ADPglucose transporter), wax (granule-bound starch synthase), isa1 (debranching enzyme isoamylase 1) and sex6 (starch synthase IIa) mutations were introgressed into NFC Tipple to give mutant and wild-type BC2F4 families with different genomic contributions of the donor parent. Comparison of starch and grain properties between the donor parents, the BC2F4 families and NFC Tipple allowed the effects of the mutations to be distinguished from genetic background effects. The wax and sex6 mutations had marked effects on starch properties regardless of genetic background. The sex6 mutation conditioned low grain weight and starch content, but the wax mutation did not. The lys5 mutation conditioned low grain weight and starch content, but exceptionally high β-glucan contents. The isa1 mutation promotes synthesis of soluble α-glucan (phytoglycogen). Its introgression into NFC Tipple increased grain weight and total α-glucan content relative to the donor parent, but reduced the ratio of phytoglycogen to starch. This study shows that introgression of mutations into a common, commercial background provides new insights that could not be gained from the donor parent. PMID:24748716

  17. Effects of granule-bound starch synthase I-defective mutation on the morphology and structure of pyrenoidal starch in Chlamydomonas.

    PubMed

    Izumo, Asako; Fujiwara, Shoko; Sakurai, Toshihiro; Ball, Steven G; Ishii, Yoshimi; Ono, Hikaru; Yoshida, Mayumi; Fujita, Naoko; Nakamura, Yasunori; Buléon, Alain; Tsuzuki, Mikio

    2011-02-01

    Lowering of the CO₂ concentration in the environment induces development of a pyrenoidal starch sheath, as well as that of pyrenoid and CO₂-concentrating mechanisms, in many microalgae. In the green algae Chlamydomonas and Chlorella, activity of granule-bound starch synthase (GBSS) concomitantly increases under these conditions. In this study, effects of the GBSS-defective mutation (sta2) on the development of pyrenoidal starch were investigated in Chlamydomonas. Stroma starch- and pyrenoid starch-enriched samples were obtained from log-phase cells grown with air containing 5% CO₂ (high-CO₂ conditions favouring stromal starch synthesis) and from those transferred to low-CO₂ conditions (air level, 0.04% CO₂, favouring pyrenoidal starch synthesis) for 6h, respectively. In the wild type, total starch content per culture volume did not increase during the low-CO₂ conditions, in spite of the development of pyrenoidal starch, suggesting that degradation of some part of stroma starch and synthesis of pyrenoid starch simultaneously occur under these conditions. Even in the GBSS-deficient mutants, pyrenoid and pyrenoid starch enlarged after lowering of the CO₂ concentration. However, the morphology of the pyrenoid starch was thinner and more fragile than the wild type, suggesting that GBSS does affect the morphology of pyrenoidal starch. Surprisingly normal GBSS activity is shown to be required to obtain the high A-type crystallinity levels that we now report for pyrenoidal starch. A model is presented explaining how GBSS-induced starch granule fusion may facilitate the formation of the pyrenoidal starch sheath.

  18. Two Additional Phosphorylases in Developing Maize Seeds 12

    PubMed Central

    Tsai, C. Y.; Nelson, O. E.

    1969-01-01

    Two additional phosphorylases (III and IV) have been detected in developing seeds of maize. Phosphorylase IV is found only in the embryo (with scutellum). It is also present in the embryo of the germinating seed where its activity is 90-fold greater than the activity in the developing embryo 22 days after pollination. Phosphorylase IV is eluted from a DEAE-cellulose column in the same fraction as phosphorylase I of the endosperm, and the 2 enzymes are similar in many respects. Phosphorylase IV is distinguished from phosphorylase I by electrophoretic mobility, by pH optimum, and because its properties are not affected by the shrunken-4 mutation. Phosphorylase III is found both in the endosperms and embryos of developing seeds. Activity for this enzyme is not detected in crude homogenates nor eluates from a DEAE-cellulose column apparently because it complexes with a non-dialyzable, heat-labile inhibitor. High activity is found after protamine sulfate fractionation. Phosphorylase III is bound to protamine sulfate and is then removed by washing with 0.3 m phosphate buffer. Phosphorylase III activity in the endosperm is not detectable 8 days after pollination but is present 12 days after pollination. Phosphorylase III differs from phosphorylases I, II, and IV in several respects—pH optimum, pH-independent ATP inhibition, time of appearance in the endosperm, and because purine and pyrimidine nucleotides are equally inhibitory. In common with phosphorylase II, phosphorylase III apparently does not require a primer to initiate the synthesis of an amylose-like polymer. PMID:5774172

  19. Registration of Common Wheat Germplasm with Mutations in SBEII Genes Conferring Increased Grain Amylose and Resistant Starch Content

    PubMed Central

    Schönhofen, André; Hazard, Brittany; Zhang, Xiaoqin; Dubcovsky, Jorge

    2016-01-01

    Starch present in the endosperm of common wheat (Triticum aestivum L.) grains is an important source of carbohydrates worldwide. Starches with a greater proportion of amylose have increased levels of resistant starch, a dietary fiber that can provide human health benefits. Induced mutations in STARCH BRANCHING ENZYME II (SBEII) genes in wheat are associated with increased amylose and resistant starch. Ethyl methane sulfonate mutations in SBEIIa and SBEIIb paralogs were combined in the hexaploid wheat cultivar Lassik. Four mutant combinations were generated: SBEIIa/b-AB (Reg. No. GP-997, PI 675644); SBEIIa/b-A, SBEIIa-D (Reg. No. GP-998, PI 675645); SBEIIa/b-B, SBEIIa-D (Reg. No. GP-999, PI 675646); and SBEIIa/b-AB, SBEIIa-D (Reg. No. GP-1000, PI 675647). The SBEII mutant lines were compared with a wild-type control in a greenhouse and field experiment. The quintuple mutant line (SBEIIa/b-AB, SBEIIa-D) presented significant increases in both amylose (51% greenhouse; 63% field) and resistant starch (947% greenhouse; 1057% field) relative to the control. A decrease in total starch content (7.8%) was observed in the field experiment. The quintuple mutant also differed in starch viscosity parameters. Registration of the hexaploid wheat SBEII-mutant lines by University of California, Davis can help expedite the development of common wheat cultivars with increased amylose and resistant starch content.

  20. Registration of Durum Wheat Germplasm Lines with Combined Mutations in SBEIIa and SBEIIb Genes Conferring Increased Amylose and Resistant Starch

    PubMed Central

    Hazard, Brittany; Zhang, Xiaoqin; Naemeh, Mahmoudreza; Dubcovsky, Jorge

    2016-01-01

    Durum wheat [Triticum turgidum L. subsp. durum (Desf.) Husn.], used in pasta, couscous, and flatbread production, is an important source of starch food products worldwide. The amylose portion of the starch forms resistant starch complexes that resist digestion and contribute to dietary fiber. Increasing the amount of amylose and resistant starch in wheat by mutating the STARCH BRANCHING ENZYME II (SBEII) genes has potential to provide human health benefits. Ethyl methane sulfonate mutations in the linked SBEIIa and SBEIIb paralogs were combined on chromosomes 2A (SBEIIa/b-A; Reg. No. GP-968, PI 670159), 2B (SBEIIa/b-B; Reg. No. GP-970, PI 670161), and on both chromosomes (SBEIIa/b-AB; Reg. No. GP-969, PI 670160) in the tetraploid wheat cultivar Kronos, a semidwarf durum wheat cultivar that has high yield potential and excellent pasta quality. These three double and quadruple SBEII-mutant lines were compared with a control sib line with no SBEII mutations in two field locations in California. The SBEIIa/b-AB line with four mutations showed dramatic increases in amylose (average 66%) and resistant starch (average 753%) relative to the control. However, the SBEIIa/b-AB line also showed an average 7% decrease in total starch and an 8% decrease in kernel weight. The release by the University of California–Davis of the durum wheat germplasm combining four SBEIIa and SBEIIb mutations will accelerate the deployment of these mutations in durum wheat breeding programs and the development of durum wheat varieties with increased resistant starch. PMID:27110322

  1. SNPs in genes functional in starch-sugar interconversion associate with natural variation of tuber starch and sugar content of potato (Solanum tuberosum L.).

    PubMed

    Schreiber, Lena; Nader-Nieto, Anna Camila; Schönhals, Elske Maria; Walkemeier, Birgit; Gebhardt, Christiane

    2014-10-01

    Starch accumulation and breakdown are vital processes in plant storage organs such as seeds, roots, and tubers. In tubers of potato (Solanum tuberosum L.) a small fraction of starch is converted into the reducing sugars glucose and fructose. Reducing sugars accumulate in response to cold temperatures. Even small quantities of reducing sugars affect negatively the quality of processed products such as chips and French fries. Tuber starch and sugar content are inversely correlated complex traits that are controlled by multiple genetic and environmental factors. Based on in silico annotation of the potato genome sequence, 123 loci are involved in starch-sugar interconversion, approximately half of which have been previously cloned and characterized. By means of candidate gene association mapping, we identified single-nucleotide polymorphisms (SNPs) in eight genes known to have key functions in starch-sugar interconversion, which were diagnostic for increased tuber starch and/or decreased sugar content and vice versa. Most positive or negative effects of SNPs on tuber-reducing sugar content were reproducible in two different collections of potato cultivars. The diagnostic SNP markers are useful for breeding applications. An allele of the plastidic starch phosphorylase PHO1a associated with increased tuber starch content was cloned as full-length cDNA and characterized. The PHO1a-HA allele has several amino acid changes, one of which is unique among all known starch/glycogen phosphorylases. This mutation might cause reduced enzyme activity due to impaired formation of the active dimers, thereby limiting starch breakdown. PMID:25081979

  2. SNPs in genes functional in starch-sugar interconversion associate with natural variation of tuber starch and sugar content of potato (Solanum tuberosum L.).

    PubMed

    Schreiber, Lena; Nader-Nieto, Anna Camila; Schönhals, Elske Maria; Walkemeier, Birgit; Gebhardt, Christiane

    2014-07-31

    Starch accumulation and breakdown are vital processes in plant storage organs such as seeds, roots, and tubers. In tubers of potato (Solanum tuberosum L.) a small fraction of starch is converted into the reducing sugars glucose and fructose. Reducing sugars accumulate in response to cold temperatures. Even small quantities of reducing sugars affect negatively the quality of processed products such as chips and French fries. Tuber starch and sugar content are inversely correlated complex traits that are controlled by multiple genetic and environmental factors. Based on in silico annotation of the potato genome sequence, 123 loci are involved in starch-sugar interconversion, approximately half of which have been previously cloned and characterized. By means of candidate gene association mapping, we identified single-nucleotide polymorphisms (SNPs) in eight genes known to have key functions in starch-sugar interconversion, which were diagnostic for increased tuber starch and/or decreased sugar content and vice versa. Most positive or negative effects of SNPs on tuber-reducing sugar content were reproducible in two different collections of potato cultivars. The diagnostic SNP markers are useful for breeding applications. An allele of the plastidic starch phosphorylase PHO1a associated with increased tuber starch content was cloned as full-length cDNA and characterized. The PHO1a-HA allele has several amino acid changes, one of which is unique among all known starch/glycogen phosphorylases. This mutation might cause reduced enzyme activity due to impaired formation of the active dimers, thereby limiting starch breakdown.

  3. Mutants of Arabidopsis with altered regulation of starch degradation

    SciTech Connect

    Caspar, T.; Lin, Tsanpiao; Kakefuda, G.; Benbow, L.; Preiss, J.; Somerville, C. )

    1991-04-01

    Mutants of Arabidopsis thaliana (L.) Heynh. with altered regulation of starch degradation were identified by screening for plants that retained high levels of leaf starch after a period of extended darkness. The mutant phenotype was also expressed in seeds, flowers, and roots, indicating that the same pathway of starch degradation is used in these tissues. In many respects, the physiological consequences of the mutations were equivalent to the effects observed in previously characterized mutants of Arabidopsis that are unable to synthesize starch. One mutant line, which was characterized in detail, had normal levels of activity of the starch degradative enzymes {alpha}-amylase, {beta}-amylase, phosphorylase, D-enzyme, and debranching enzyme. Thus, it was not possible to establish a biochemical basis for the phenotype, which was due to a recessive mutant at a locus designated sex 1 at position 12.2 on chromosome 1. This raises the possibility that hitherto unidentified factors, altered by the mutation, play a key role in regulating or catalyzing starch degradation.

  4. The Crystal Structure of Streptococcus pyogenes Uridine Phosphorylase Reveals a Distinct Subfamily of Nucleoside Phosphorylases

    SciTech Connect

    Tran, Timothy H.; Christoffersen, S.; Allan, Paula W.; Parker, William B.; Piskur, Jure; Serra, I.; Terreni, M.; Ealick, Steven E.

    2011-09-20

    Uridine phosphorylase (UP), a key enzyme in the pyrimidine salvage pathway, catalyzes the reversible phosphorolysis of uridine or 2'-deoxyuridine to uracil and ribose 1-phosphate or 2'-deoxyribose 1-phosphate. This enzyme belongs to the nucleoside phosphorylase I superfamily whose members show diverse specificity for nucleoside substrates. Phylogenetic analysis shows Streptococcus pyogenes uridine phosphorylase (SpUP) is found in a distinct branch of the pyrimidine subfamily of nucleoside phosphorylases. To further characterize SpUP, we determined the crystal structure in complex with the products, ribose 1-phosphate and uracil, at 1.8 {angstrom} resolution. Like Escherichia coli UP (EcUP), the biological unit of SpUP is a hexamer with an ?/? monomeric fold. A novel feature of the active site is the presence of His169, which structurally aligns with Arg168 of the EcUP structure. A second active site residue, Lys162, is not present in previously determined UP structures and interacts with O2 of uracil. Biochemical studies of wild-type SpUP showed that its substrate specificity is similar to that of EcUP, while EcUP is {approx}7-fold more efficient than SpUP. Biochemical studies of SpUP mutants showed that mutations of His169 reduced activity, while mutation of Lys162 abolished all activity, suggesting that the negative charge in the transition state resides mostly on uracil O2. This is in contrast to EcUP for which transition state stabilization occurs mostly at O4.

  5. [Purine nucleoside phosphorylase].

    PubMed

    Pogosian, L G; Akopian, Zh I

    2013-01-01

    Purine nucleoside phosphorylase (PNP) is one of the most important enzymes of the purine metabolism, wich promotes the recycling of purine bases. Nowadays is the actual to search for effective inhibitors of this enzyme which is necessary for creation T-cell immunodeficient status of the organism in the organs and tissues transplantation, and chemotherapy of a number pathologies as well. For their successful practical application necessary to conduct in-depth and comprehensive study of the enzyme, namely a structure, functions, and an affinity of the reaction mechanism. In the review the contemporary achievements in the study of PNP from various biological objects are presented. New data describing the structure of PNP are summarised and analysed. The physiological role of the enzyme is discussed. The enzyme basic reaction mechanisms and actions are considered. The studies on enzyme physicochemical, kinetic, and catalytic research are presented. PMID:24479338

  6. Role of phosphorylase in the mechanism of potato minituber storage cell changes during clinorotation

    NASA Astrophysics Data System (ADS)

    Nedukha, O.; Shnyukova, E.

    The differences between the cytochemical reaction intensity and activity of phosphorylase (EC 2.4.1.1) and carbohydrate content in storage parenchyma cells of Solanum tuberosum L. (cv Adreta) minitubers grown for 30 days in the horizontal clinostate (2 rev/min) and in the control have been studied by electroncytochemical and biochemical methods. It is established an acceleration of minitubers formation and storage parenchyma cell differentiation at clinorotation. Electroncytochemical investigation of phosphorylase activity localization in the storage parenchyma cells of minitubers grown in control and at clinorotation showed the product of the reaction as electron-dense precipitate was marked plastids. Intensity and density of precipitate was increase in stroma of plastids and on starch grain surface during of intensive growth of starch in amyloplast (on 10- and 20-days of the minituber formation) of clinorotated minitubers in comparison with that in the control. The precipitate amount was decreased in the plastids on 30 day of growth in both variants. Using biochemical methods it is found that activity of phosphorylase and content of mono- and disaccharide and also starch content changed in minitubers formed during clinorotation and in the control. Data obtained are discussed regarding the possible mechanism of phosphorylase activity change and the role of mono- and disaccharide in acceleration of storage organ formation during clinorotation.

  7. Characterization of a starch-hydrolyzing α-amylase produced by Aspergillus niger WLB42 mutated by ethyl methanesulfonate treatment

    PubMed Central

    Wang, Shihui; Lin, Chaoyang; Liu, Yun; Shen, Zhicheng; Jeyaseelan, Jenasia; Qin, Wensheng

    2016-01-01

    Aspergillus niger is the most commonly used fungus for commercial amylase production, the increase of amylase activity will be beneficial to the amylase industry. Herein we report a high α-amylase producing (HAP) A. niger WLB42 mutated from A. niger A4 by ethyl methanesulfonate treatment. The fermentation conditions for the amylase production were optimized. The results showed that both the amylase activity and total protein content reached highest after 48-h incubation in liquid medium using starch as the sole carbon source. The enzyme production reached maximum at temperature of 30°C, pH 7, with 40 g/L starch in the medium inoculated with 1.4% v/v spore. When 0.3% w/v urea was added to the liquid medium as a nitrogen source, the amylase activity was elevated by 20%. Nine monosaccharides and derivatives were tested for α-amylase induction, glucose was the best inducer. Furthermore, the enzymology characterization of amylase was conducted. The molecular weight of amylase was determined to be 50 kD by SDS-PAGE. The amylase had maximum activity at 45°C and pH 7. The activity could be dramatically triggered by adding 1 mM Co2+, increased to 250%. The activity was inhibited by detergents SDS and Triton X-100. Six different brands of starch were tested for amylase activity, the results demonstrated that the more soluble of the starch, the higher hydrolyzability of the substrate by amylase. PMID:27335681

  8. Characterization of a starch-hydrolyzing α-amylase produced by Aspergillus niger WLB42 mutated by ethyl methanesulfonate treatment.

    PubMed

    Wang, Shihui; Lin, Chaoyang; Liu, Yun; Shen, Zhicheng; Jeyaseelan, Jenasia; Qin, Wensheng

    2016-01-01

    Aspergillus niger is the most commonly used fungus for commercial amylase production, the increase of amylase activity will be beneficial to the amylase industry. Herein we report a high α-amylase producing (HAP) A. niger WLB42 mutated from A. niger A4 by ethyl methanesulfonate treatment. The fermentation conditions for the amylase production were optimized. The results showed that both the amylase activity and total protein content reached highest after 48-h incubation in liquid medium using starch as the sole carbon source. The enzyme production reached maximum at temperature of 30°C, pH 7, with 40 g/L starch in the medium inoculated with 1.4% v/v spore. When 0.3% w/v urea was added to the liquid medium as a nitrogen source, the amylase activity was elevated by 20%. Nine monosaccharides and derivatives were tested for α-amylase induction, glucose was the best inducer. Furthermore, the enzymology characterization of amylase was conducted. The molecular weight of amylase was determined to be 50 kD by SDS-PAGE. The amylase had maximum activity at 45°C and pH 7. The activity could be dramatically triggered by adding 1 mM Co(2+), increased to 250%. The activity was inhibited by detergents SDS and Triton X-100. Six different brands of starch were tested for amylase activity, the results demonstrated that the more soluble of the starch, the higher hydrolyzability of the substrate by amylase. PMID:27335681

  9. Evaluation of novel starch-deficient mutants of Chlorella sorokiniana for hyper-accumulation of lipids

    PubMed Central

    Vonlanthen, Sofie; Dauvillée, David; Purton, Saul

    2015-01-01

    When green algae are exposed to physiological stresses such as nutrient deprivation, growth is arrested and the cells channel fixed carbon instead into storage compounds, accumulating first starch granules and then lipid bodies containing triacylglycerides. In recent years there has been significant interest in the commercial exploitation of algal lipids as a sustainable source of biodiesel. Since starch and lipid biosynthesis involves the same C3 precursor pool, it has been proposed that mutations blocking starch accumulation should result in increased lipid yields, and indeed several studies have supported this. The fast-growing, thermotolerant alga Chlorella sorokiniana represents an attractive strain for industrial cultivation. We have therefore generated and characterized starch-deficient mutants of C. sorokiniana and determined whether lipid levels are increased in these strains under stress conditions. One mutant (ST68) is shown to lack isoamylase, whilst two others (ST3 and ST12) are defective in starch phosphorylase. However, we find no significant change in the accumulation or profile of fatty acids in these mutants compared to the wild-type, suggesting that a failure to accumulate starch per se is not sufficient for the hyper-accumulation of lipid, and that more subtle regulatory steps underlie the partitioning of carbon to the two storage products. PMID:26865991

  10. Starch Degradation in the Cotyledons of Germinating Lentils

    PubMed Central

    Tárrago, Jorge Fernández; Nicolás, Gregorio

    1976-01-01

    Starch, total amylolytic and phosphorylase activities were determined in lentil cotyledons during the first days of germination. Several independent criteria show that the amylolytic activity is due mainly to an amylase of the α type. Starch is degraded slowly in the first days; during this time, α- and β-amylase activity are very low, while phosphorylase increases and reach a peak on the 3rd day. On the 4th day, there is a more rapid depletion of starch which coincides with an increase in α-amylase activity. By polyacrylamide gel electrophoresis of the crude starch-degrading enzyme, five bands were obtained: one phosphorylase, three α-amylases, and one β-amylase. Based on their heat lability or heat stability, two sets of α-amylase seem to exist in lentil cotyledons. Images PMID:16659730

  11. Fetal-onset severe skeletal muscle glycogenosis associated with phosphorylase-b kinase deficiency.

    PubMed

    Bührer, C; van Landeghem, F; Brück, W; Felderhoff-Müser, U; Vorgerd, M; Obladen, M

    2000-04-01

    We report on a premature newborn girl delivered after 32 weeks of gestation by cesarean section after sparse limb movements, fetal tachycardia and late heart rate decelerations had suggested fetal distress. Following 1 day of mechanical ventilation, adequate pulmonary gas exchange was achieved by spontaneous breathing. Main symptoms were virtually complete absence of spontaneous movements, increased flexor tonus of the extremities, and hypotonia of the trunk. Inability to suck or swallow required nasogastric gavage feeding. There were no hypoglycemic episodes. Echocardiography revealed normal myocardial function. Creatine kinase was 237 U/I at 2 days of life, declining to normal values thereafter. Muscle biopsy revealed increased glycogen storage with subsarcolemmal glycogen deposits and low phosphorylase-a activity while total phosphorylase was normal after in vitro activation, suggestive of phosphorylase-b kinase deficiency. No mutation was detected in exon 1 of the myophosphorylase gene. No psychomotor development was observed, and the infant died of central apnea at 3 months of age.

  12. A Putative Gene sbe3-rs for Resistant Starch Mutated from SBE3 for Starch Branching Enzyme in Rice (Oryza sativa L.)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Foods high in resistant starch (RS) are beneficial to prevent various diseases including diabetes, colon cancer, diarrhea and chronic renal or hepatic diseases. Elevated RS in rice is important for public health since rice is a staple food for half of the world’s population. A japonica mutant ‘Jiang...

  13. Glycal Formation in Crystals of Uridine Phosphorylase

    SciTech Connect

    Paul, Debamita; O’Leary, Sen E.; Rajashankar, Kanagalaghatta; Bu, Weiming; Toms, Angela; Settembre, Ethan C.; Sanders, Jennie M.; Begley, Tadhg P.; Ealick, Steven E.

    2010-06-22

    Uridine phosphorylase is a key enzyme in the pyrimidine salvage pathway. This enzyme catalyzes the reversible phosphorolysis of uridine to uracil and ribose 1-phosphate (or 2{prime}-deoxyuridine to 2{prime}-deoxyribose 1-phosphate). Here we report the structure of hexameric Escherichia coli uridine phosphorylase treated with 5-fluorouridine and sulfate and dimeric bovine uridine phosphorylase treated with 5-fluoro-2{prime}-deoxyuridine or uridine, plus sulfate. In each case the electron density shows three separate species corresponding to the pyrimidine base, sulfate, and a ribosyl species, which can be modeled as a glycal. In the structures of the glycal complexes, the fluorouracil O2 atom is appropriately positioned to act as the base required for glycal formation via deprotonation at C2{prime}. Crystals of bovine uridine phosphorylase treated with 2{prime}-deoxyuridine and sulfate show intact nucleoside. NMR time course studies demonstrate that uridine phosphorylase can catalyze the hydrolysis of the fluorinated nucleosides in the absence of phosphate or sulfate, without the release of intermediates or enzyme inactivation. These results add a previously unencountered mechanistic motif to the body of information on glycal formation by enzymes catalyzing the cleavage of glycosyl bonds.

  14. Enzymatic transformation of nonfood biomass to starch.

    PubMed

    You, Chun; Chen, Hongge; Myung, Suwan; Sathitsuksanoh, Noppadon; Ma, Hui; Zhang, Xiao-Zhou; Li, Jianyong; Zhang, Y-H Percival

    2013-04-30

    The global demand for food could double in another 40 y owing to growth in the population and food consumption per capita. To meet the world's future food and sustainability needs for biofuels and renewable materials, the production of starch-rich cereals and cellulose-rich bioenergy plants must grow substantially while minimizing agriculture's environmental footprint and conserving biodiversity. Here we demonstrate one-pot enzymatic conversion of pretreated biomass to starch through a nonnatural synthetic enzymatic pathway composed of endoglucanase, cellobiohydrolyase, cellobiose phosphorylase, and alpha-glucan phosphorylase originating from bacterial, fungal, and plant sources. A special polypeptide cap in potato alpha-glucan phosphorylase was essential to push a partially hydrolyzed intermediate of cellulose forward to the synthesis of amylose. Up to 30% of the anhydroglucose units in cellulose were converted to starch; the remaining cellulose was hydrolyzed to glucose suitable for ethanol production by yeast in the same bioreactor. Next-generation biorefineries based on simultaneous enzymatic biotransformation and microbial fermentation could address the food, biofuels, and environment trilemma.

  15. Enzymatic transformation of nonfood biomass to starch

    PubMed Central

    You, Chun; Chen, Hongge; Myung, Suwan; Sathitsuksanoh, Noppadon; Ma, Hui; Zhang, Xiao-Zhou; Li, Jianyong; Zhang, Y.-H. Percival

    2013-01-01

    The global demand for food could double in another 40 y owing to growth in the population and food consumption per capita. To meet the world’s future food and sustainability needs for biofuels and renewable materials, the production of starch-rich cereals and cellulose-rich bioenergy plants must grow substantially while minimizing agriculture’s environmental footprint and conserving biodiversity. Here we demonstrate one-pot enzymatic conversion of pretreated biomass to starch through a nonnatural synthetic enzymatic pathway composed of endoglucanase, cellobiohydrolyase, cellobiose phosphorylase, and alpha-glucan phosphorylase originating from bacterial, fungal, and plant sources. A special polypeptide cap in potato alpha-glucan phosphorylase was essential to push a partially hydrolyzed intermediate of cellulose forward to the synthesis of amylose. Up to 30% of the anhydroglucose units in cellulose were converted to starch; the remaining cellulose was hydrolyzed to glucose suitable for ethanol production by yeast in the same bioreactor. Next-generation biorefineries based on simultaneous enzymatic biotransformation and microbial fermentation could address the food, biofuels, and environment trilemma. PMID:23589840

  16. Starch poisoning

    MedlinePlus

    Cooking starch poisoning; Laundry starch poisoning ... Cooking and laundry starch are both made from vegetable products, most commonly: Corn Potatoes Rice Wheat Both are usually considered nonpoisonous (nontoxic), but ...

  17. Soybean cotyledon starch metabolism is sensitive to altered gravity conditions

    NASA Technical Reports Server (NTRS)

    Brown, C. S.; Piastuch, W. C.; Knott, W. M.

    1994-01-01

    We have demonstrated that etiolated soybean seedlings grown under the altered gravity conditions of clinorotation (1 rpm) and centrifugation (5xg) exhibit changes in starch metabolism. Cotyledon starch concentration was lower (-28%) in clinorotated plants and higher (+24%) in centrifuged plants than in vertical control plants. The activity of ADP-glucose pyrophosphorylase in the cotyledons was affected in a similar way, i.e. lower (-37%) in the clinorotated plants and higher (+22%) in the centrifuged plants. Other starch metabolic enzyme activities, starch synthase, starch phosphorylase and total hydrolase were not affected by the altered gravity treatments. We conclude that the observed changes in starch concentrations were primarily due to gravity-mediated differences in ADP-glucose pyrophosphorylase activity.

  18. The crystal structure and activity of a putative trypanosomal nucleoside phosphorylase reveal it to be a homodimeric uridine phosphorylase

    PubMed Central

    Larson, Eric T.; Mudeppa, Devaraja G.; Gillespie, J. Robert; Mueller, Natascha; Napuli, Alberto J.; Arif, Jennifer A.; Ross, Jenni; Arakaki, Tracy L.; Lauricella, Angela; DeTitta, George; Luft, Joseph; Zucker, Frank; Verlinde, Christophe L. M. J.; Fan, Erkang; Van Voorhis, Wesley C.; Buckner, Frederick S.; Rathod, Pradipsinh K.; Hol, Wim G. J.; Merritt, Ethan A.

    2010-01-01

    Purine nucleoside phosphorylases and uridine phosphorylases are closely related enzymes involved in purine and pyrimidine salvage, respectively, which catalyze the removal of the ribosyl moiety from nucleosides so that the nucleotide base may be recycled. Parasitic protozoa generally are incapable of de novo purine biosynthesis so the purine salvage pathway is of potential therapeutic interest. Information about pyrimidine biosynthesis in these organisms is much more limited. Though all seem to carry at least a subset of enzymes from each pathway, the dependency on de novo pyrimidine synthesis versus salvage varies from organism to organism and even from one growth stage to another. We have structurally and biochemically characterized a putative nucleoside phosphorylase from the pathogenic protozoan Trypanosoma brucei and find that it is a homodimeric uridine phosphorylase. This is the first characterization of a uridine phosphorylase from a trypanosomal source despite this activity being observed decades ago. Although this gene was broadly annotated as a putative nucleoside phosphorylase, it was widely inferred to be a purine nucleoside phosphorylase. Our characterization of this trypanosomal enzyme shows that it is possible to distinguish between purine and uridine phosphorylase activity at the sequence level based on the absence or presence of a characteristic uridine phosphorylase-specificity insert. We suggest that this recognizable feature may aid in proper annotation of the substrate specificity of enzymes in the nucleoside phosphorylase family. PMID:20070944

  19. Purine nucleoside phosphorylase polymorphism in the genus Littorina (Prosobranchia: Mollusca).

    PubMed

    Knight, A J; Ward, R D

    1986-06-01

    Examination of eight Atlantic species of the genus Littorina by starch gel electrophoresis of purine nucleoside phosphorylase revealed extensive polymorphism within the L. saxatilis complex. In this group, four alleles have been identified. Heterozygotes are four banded, and thus, as in vertebrates, the enzyme is likely to be a trimer. Breeding experiments confirmed the genetic interpretation of the phenotype patterns. Where species of the saxatilis complex [L. saxatilis (=L. rudis), L. arcana, L. nigrolineata, L. neglecta] are sympatric, there are sometimes significant allele frequency differences between them. A fifth allele was present at a high frequency in L. obtusata and L. mariae, and L. littorea and L. neritoides each possessed unique alleles. A total of eight alleles was identified. Densitometric scanning of heterozygote patterns pointed to activity differences between alleles and also showed that, while the heterotrimeric bands were never less intense than the homotrimeric bands, the heterotrimeric bands were sometimes less intense than expected. It is not clear whether this represents nonrandom association of subunits, decreased stability of heterotrimers, or simply an artifact of the staining and quantifying process. PMID:3091000

  20. Myoglobinuria and Skeletal Muscle Phosphorylase Deficiency

    PubMed Central

    Nixon, J. C.; Hobbs, W. K.; Greenblatt, J.

    1966-01-01

    Investigation of a patient complaining of exercise-induced dark urine, pain, stiffness and tenderness of skeletal muscle revealed findings characteristic of McArdle's disease. The dark urine was attributable to the excretion of myoglobin, and an ischemic exercise test failed to demonstrate the usual rise and fall in blood lactate and pyruvate. Enzyme assays of skeletal muscle showed an absence of phosphorylase, a slight increase in phosphorylase b kinase and a slight decrease in phosphoglucomutase. Chemical and histochemical analyses demonstrated an increase in the skeletal muscle glycogen content and an enlargement of the muscle cells. No abnormality of liver glycogen metabolism was found. In the absence of specific therapy, an effective and practical form of treatment is reduction of exercise below the threshold of symptoms. ImagesFig. 1Fig. 2Fig. 6Fig. 7Fig. 8 PMID:4952390

  1. Plantain and banana starches: granule structural characteristics explain the differences in their starch degradation patterns.

    PubMed

    Soares, Claudinéia Aparecida; Peroni-Okita, Fernanda Helena Gonçalves; Cardoso, Mateus Borba; Shitakubo, Renata; Lajolo, Franco Maria; Cordenunsi, Beatriz Rosana

    2011-06-22

    Different banana cultivars were used to investigate the influences of starch granule structure and hydrolases on degradation. The highest degrees of starch degradation were observed in dessert bananas during ripening. Scanning electron microscopy images revealed smooth granule surface in the green stage in all cultivars, except for Mysore. The small and round granules were preferentially degraded in all of the cultivars. Terra demonstrated a higher degree of crystallinity and a short amylopectin chain length distribution, resulting in high starch content in the ripe stage. Amylose content and the crystallinity index were more strongly correlated than the distribution of amylopectin branch chain lengths in banana starches. α- and β-amylase activities were found in both forms, soluble in the pulp and associated with the starch granule. Starch-phosphorylase was not found in Mysore. On the basis of the profile of α-amylase in vitro digestion and the structural characteristics, it could be concluded that the starch of plantains has an arrangement of granules more resistant to enzymes than the starch of dessert bananas. PMID:21591784

  2. Plantain and banana starches: granule structural characteristics explain the differences in their starch degradation patterns.

    PubMed

    Soares, Claudinéia Aparecida; Peroni-Okita, Fernanda Helena Gonçalves; Cardoso, Mateus Borba; Shitakubo, Renata; Lajolo, Franco Maria; Cordenunsi, Beatriz Rosana

    2011-06-22

    Different banana cultivars were used to investigate the influences of starch granule structure and hydrolases on degradation. The highest degrees of starch degradation were observed in dessert bananas during ripening. Scanning electron microscopy images revealed smooth granule surface in the green stage in all cultivars, except for Mysore. The small and round granules were preferentially degraded in all of the cultivars. Terra demonstrated a higher degree of crystallinity and a short amylopectin chain length distribution, resulting in high starch content in the ripe stage. Amylose content and the crystallinity index were more strongly correlated than the distribution of amylopectin branch chain lengths in banana starches. α- and β-amylase activities were found in both forms, soluble in the pulp and associated with the starch granule. Starch-phosphorylase was not found in Mysore. On the basis of the profile of α-amylase in vitro digestion and the structural characteristics, it could be concluded that the starch of plantains has an arrangement of granules more resistant to enzymes than the starch of dessert bananas.

  3. Modified 5-fluorouracil: Uridine phosphorylase inhibitor

    NASA Astrophysics Data System (ADS)

    Lashkov, A. A.; Shchekotikhin, A. A.; Shtil, A. A.; Sotnichenko, S. E.; Mikhailov, A. M.

    2016-09-01

    5-Fluorouracil (5-FU) is a medication widely used in chemotherapy to treat various types of cancer. Being a substrate for the reverse reaction catalyzed by uridine phosphorylase (UPase), 5-FU serves as a promising prototype molecule (molecular scaffold) for the design of a selective UPase inhibitor that enhances the antitumor activity of 5-FU and exhibits intrinsic cytostatic effects on cancer cells. The chemical formula of the new compound, which binds to the uracil-binding site and, in the presence of a phosphate anion, to the phosphate-binding site of UPase, is proposed and investigated by molecular simulation methods.

  4. Glucose 1-phosphate is efficiently taken up by potato (Solanum tuberosum) tuber parenchyma cells and converted to reserve starch granules.

    PubMed

    Fettke, Joerg; Albrecht, Tanja; Hejazi, Mahdi; Mahlow, Sebastian; Nakamura, Yasunori; Steup, Martin

    2010-02-01

    Reserve starch is an important plant product but the actual biosynthetic process is not yet fully understood. Potato (Solanum tuberosum) tuber discs from various transgenic plants were used to analyse the conversion of external sugars or sugar derivatives to starch. By using in vitro assays, a direct glucosyl transfer from glucose 1-phosphate to native starch granules as mediated by recombinant plastidial phosphorylase was analysed. Compared with labelled glucose, glucose 6-phosphate or sucrose, tuber discs converted externally supplied [(14)C]glucose 1-phosphate into starch at a much higher rate. Likewise, tuber discs from transgenic lines with a strongly reduced expression of cytosolic phosphoglucomutase, phosphorylase or transglucosidase converted glucose 1-phosphate to starch with the same or even an increased rate compared with the wild-type. Similar results were obtained with transgenic potato lines possessing a strongly reduced activity of both the cytosolic and the plastidial phosphoglucomutase. Starch labelling was, however, significantly diminished in transgenic lines, with a reduced concentration of the plastidial phosphorylase isozymes. Two distinct paths of reserve starch biosynthesis are proposed that explain, at a biochemical level, the phenotype of several transgenic plant lines.

  5. Regulation of synthase phosphatase and phosphorylase phosphatase in rat liver.

    PubMed

    Tan, A W; Nuttall, F Q

    1976-08-12

    Using substrates purified from liver, the apparent Km values of synthase phosphatase ([UDPglucose--glycogen glucosyltransferase-D]phosphohydrolase, EC 3.1.3.42) and phosphorylase phosphatase (phosphorylase a phosphohydrolase, EC 3.1.3.17) were found to be 0.7 and 60 units/ml respectively. The maximal velocity of phosphorylase phosphatase was more than a 100 times that of synthase phosphatase. In adrenalectomized, fasted animals there was a complete loss of synthase phosphatase but only a slight decrease in phosphorylase phosphatase when activity was measured using endogenous substrates in a concentrated liver extract. When assayed under optimal conditions with purified substrates, both activities were present but had decreased to very low levels. Mixing experiments indicated that synthase D present in the extract of adrenalectomized fasted animals was altered such that it was no longer a substrate for synthase phosphatase from normal rats. Phosphorylase a substrate on the other hand was unaltered and readily converted. When glucose was given in vivo, no change in percent of synthase in the I form was seen in adrenalectomized rats but the percent of phosphorylase in the a form was reduced. Precipitation of protein from an extract of normal fed rats with ethanol produced a large activation of phosphorylase phosphatase activity with no corresponding increase in synthase phosphatase activity. Despite the low phosphorylase phosphatase present in extracts of adrenalectomized fasted animals, ethanol precipitation increased activity to the same high level as obtained in the normal fed rats. Synthase phosphatase and phosphorylase phosphatase activities were also decreased in normal fasted, diabetic fed and fasted, and adrenalectomized fed rats. Both enzymes recovered in the same manner temporally after oral glucose administration to adrenalectomized, fasted rats. These results suggest an integrated regulatory mechanism for the two phosphatase.

  6. Structural analyses reveal two distinct families of nucleoside phosphorylases.

    PubMed Central

    Pugmire, Matthew J; Ealick, Steven E

    2002-01-01

    The reversible phosphorolysis of purine and pyrimidine nucleosides is an important biochemical reaction in the salvage pathway, which provides an alternative to the de novo purine and pyrimidine biosynthetic pathways. Structural studies in our laboratory and by others have revealed that only two folds exist that catalyse the phosphorolysis of all nucleosides, and provide the basis for defining two families of nucleoside phosphorylases. The first family (nucleoside phosphorylase-I) includes enzymes that share a common single-domain subunit, with either a trimeric or a hexameric quaternary structure, and accept a range of both purine and pyrimidine nucleoside substrates. Despite differences in substrate specificity, amino acid sequence and quaternary structure, all members of this family share a characteristic subunit topology. We have also carried out a sequence motif study that identified regions of the common subunit fold that are functionally significant in differentiating the various members of the nucleoside phosphorylase-I family. Although the substrate-binding sites are arranged similarly for all members of the nucleoside phosphorylase-I family, a comparison of the active sites from the known structures of this family indicates significant differences between the trimeric and hexameric family members. Sequence comparisons also suggest structural identity between the nucleoside phosphorylase-I family and both 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase and AMP nucleosidase. Members of the second family of nucleoside phosphorylases (nucleoside phosphorylase-II) share a common two-domain subunit fold and a dimeric quaternary structure, share a significant level of sequence identity (>30%) and are specific for pyrimidine nucleosides. Members of this second family accept both thymidine and uridine substrates in lower organisms, but are specific for thymidine in mammals and other higher organisms. A possible relationship between nucleoside

  7. [Enzymes of starch metabolism in the chloroplasts of the bundle sheath and palisade cells of Zea mays].

    PubMed

    Huber, W; de Fekete, M A; Ziegler, H

    1969-12-01

    Isolated chloroplasts from the bundle sheath cells show considerable activity of the ADPG- and UDPG-pyrophosphorylase (EC 2.7.7.9), ADPG- and UDPG-transglucosylase (EC 2.4.1.21), and the starch phosphorylase (EC 2.4.1.1). In chloroplasts of the palisade cells, on the other hand, only the UDPG-pyrophosphorylase is remarkably active.

  8. Immobilized phosphorylase for synthesis of polysaccharides from glucose

    NASA Technical Reports Server (NTRS)

    Marshall, D. L.

    1972-01-01

    Continuous processes for enzymatic production of carbohydrates from glucose are discussed. Key reactant in process is identified as phosphorylase which catalyzes reversible formation or degradation of polysaccharide. Chemical compounds and reactions to synthesize polysaccharides are analyzed.

  9. Liver glycogen storage diseases due to phosphorylase system deficiencies: diagnosis thanks to non invasive blood enzymatic and molecular studies.

    PubMed

    Davit-Spraul, Anne; Piraud, Monique; Dobbelaere, Dries; Valayannopoulos, Vassili; Labrune, Philippe; Habes, Dalila; Bernard, Olivier; Jacquemin, Emmanuel; Baussan, Christiane

    2011-01-01

    Glycogen storage disease (GSD) due to a deficient hepatic phosphorylase system defines a genetically heterogeneous group of disorders that mainly manifests in children. We investigated 45 unrelated children in whom a liver GSD VI or IX was suspected on the basis of clinical symptoms including hepatomegaly, increased serum transaminases, postprandial lactatemia and/or mild fasting hypoglycemia. Liver phosphorylase and phosphorylase b kinase activities studied in peripheral blood cells allowed to suspect diagnosis in 37 cases but was uninformative in 5. Sequencing of liver phosphorylase genes was useful to establish an accurate diagnosis. Causative mutations were found either in the PYGL (11 patients), PHKA2 (26 patients), PHKG2 (three patients) or in the PHKB (three patients) genes. Eleven novel disease causative mutations, five missense (p.N188K, p.D228Y, p.P382L, p.R491H, p.L500R) and six truncating mutations (c.501_502ins361pb, c.528+2T>C, c.856-29_c.1518+614del, c.1620+1G>C, p.E703del and c.2313-1G>T) were identified in the PYGL gene. Seventeen novel disease causative mutations, ten missense (p.A42P, p.Q95R, p.G131D, p.G131V, p.Q134R, p.G187R, p.G300V, p.G300A, p.C326Y, p.W820G) and seven truncating (c.537+5G>A, p.G396DfsX28, p.Q404X, p.N653X, p.L855PfsX87, and two large deletions) were identified in the PHKA2 gene. Four novel truncating mutations (p.R168X, p.Q287X, p.I268PfsX12 and c.272-1G>C) were identified in the PHKG2 gene and three (c.573_577del, p.R364X, c.2427+3A>G) in the PHKB gene. Patients with PHKG2 mutations evolved towards cirrhosis. Molecular analysis of GSD VI or IX genes allows to confirm diagnosis suspected on the basis of enzymatic analysis and to establish diagnosis and avoid liver biopsy when enzymatic studies are not informative in blood cells.

  10. Black leaf streak disease affects starch metabolism in banana fruit.

    PubMed

    Saraiva, Lorenzo de Amorim; Castelan, Florence Polegato; Shitakubo, Renata; Hassimotto, Neuza Mariko Aymoto; Purgatto, Eduardo; Chillet, Marc; Cordenunsi, Beatriz Rosana

    2013-06-12

    Black leaf streak disease (BLSD), also known as black sigatoka, represents the main foliar disease in Brazilian banana plantations. In addition to photosynthetic leaf area losses and yield losses, this disease causes an alteration in the pre- and postharvest behavior of the fruit. The aim of this work was to investigate the starch metabolism of fruits during fruit ripening from plants infected with BLSD by evaluating carbohydrate content (i.e., starch, soluble sugars, oligosaccharides, amylose), phenolic compound content, phytohormones, enzymatic activities (i.e., starch phosphorylases, α- and β-amylase), and starch granules. The results indicated that the starch metabolism in banana fruit ripening is affected by BLSD infection. Fruit from infested plots contained unusual amounts of soluble sugars in the green stage and smaller starch granules and showed a different pattern of superficial degradation. Enzymatic activities linked to starch degradation were also altered by the disease. Moreover, the levels of indole-acetic acid and phenolic compounds indicated an advanced fruit physiological age for fruits from infested plots. PMID:23692371

  11. Thymidine phosphorylase expression in Kaposi sarcoma.

    PubMed Central

    Dada, M A; Boshoff, C H; Comley, M A; Turley, H; Schneider, J W; Chetty, R; Gatter, K C

    1996-01-01

    AIMS: To examine the immunohistochemical distribution of thymidine phosphorylase (TP) in all clinicopathological subtypes of Kaposi sarcoma. METHODS: Thirty two biopsy specimens of Kaposi sarcoma (29 patients) were studied. Six of these patients represented classic, six endemic, eight HIV associated, seven post-immunosuppression/transplant related, and two unclassified variants of Kaposi sarcoma. The average age was 49 years (range 22-83 years) and the male: female ratio 24:5. Four samples of angiosarcoma and one of spindle cell haemangio-endothelioma were stained in parallel. All specimens were fixed in formalin, embedded in paraffin wax and processed routinely. Immunohistochemistry was carried out using an antibody directed against CD31 (JC70) and the monoclonal antibody P-GF.44C against TP. RESULTS: All biopsy specimens showed immunoexpression for TP. The spindle cell component stained more strongly than newly formed endothelium lined vessels and normal, resident vessels at a distance from the lesions. CONCLUSIONS: The strong immunoexpression of TP suggests up-regulation of TP and a role for TP in angiogensis in Kaposi sarcoma. The mechanism for the up-regulation of TP remains unknown, but viral infections may trigger it. The differential staining of the various cell components of Kaposi sarcoma also suggest that TP either plays a role in the differentiation and maturation of Kaposi sarcoma or is a reflection of such changes. Images PMID:8707955

  12. Starch synthesis in Arabidopsis. Granule synthesis, composition, and structure.

    PubMed

    Zeeman, Samuel C; Tiessen, Axel; Pilling, Emma; Kato, K Lisa; Donald, Athene M; Smith, Alison M

    2002-06-01

    The aim of this work was to characterize starch synthesis, composition, and granule structure in Arabidopsis leaves. First, the potential role of starch-degrading enzymes during starch accumulation was investigated. To discover whether simultaneous synthesis and degradation of starch occurred during net accumulation, starch was labeled by supplying (14)CO(2) to intact, photosynthesizing plants. Release of this label from starch was monitored during a chase period in air, using different light intensities to vary the net rate of starch synthesis. No release of label was detected unless there was net degradation of starch during the chase. Similar experiments were performed on a mutant line (dbe1) that accumulates the soluble polysaccharide, phytoglycogen. Label was not released from phytoglycogen during the chase indicating that, even when in a soluble form, glucan is not appreciably degraded during accumulation. Second, the effect on starch composition of growth conditions and mutations causing starch accumulation was studied. An increase in starch content correlated with an increased amylose content of the starch and with an increase in the ratio of granule-bound starch synthase to soluble starch synthase activity. Third, the structural organization and morphology of Arabidopsis starch granules was studied. The starch granules were birefringent, indicating a radial organization of the polymers, and x-ray scatter analyses revealed that granules contained alternating crystalline and amorphous lamellae with a periodicity of 9 nm. Granules from the wild type and the high-starch mutant sex1 were flattened and discoid, whereas those of the high-starch mutant sex4 were larger and more rounded. These larger granules contained "growth rings" with a periodicity of 200 to 300 nm. We conclude that leaf starch is synthesized without appreciable turnover and comprises similar polymers and contains similar levels of molecular organization to storage starches, making Arabidopsis

  13. Creation of a high-amylose durum wheat through mutagenesis of starch synthase II (SSIIa)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In cereal seeds mutations in one or more starch synthases lead to decreased amylopectin and increased amylose content. Here, the impact of starch synthase IIa (SSIIa or SGP-1) mutations upon durum starch was investigated. A screen of durum accessions identified two lines lacking SGP-A1, the A geno...

  14. Activation of Phosphorylase Kinase by Physiological Temperature.

    PubMed

    Herrera, Julio E; Thompson, Jackie A; Rimmer, Mary Ashley; Nadeau, Owen W; Carlson, Gerald M

    2015-12-29

    In the six decades since its discovery, phosphorylase kinase (PhK) from rabbit skeletal muscle has usually been studied at 30 °C; in fact, not a single study has examined functions of PhK at a rabbit's body temperature, which is nearly 10 °C greater. Thus, we have examined aspects of the activity, regulation, and structure of PhK at temperatures between 0 and 40 °C. Between 0 and 30 °C, the activity at pH 6.8 of nonphosphorylated PhK predictably increased; however, between 30 and 40 °C, there was a dramatic jump in its activity, resulting in the nonactivated enzyme having a far greater activity at body temperature than was previously realized. This anomalous change in properties between 30 and 40 °C was observed for multiple functions, and both stimulation (by ADP and phosphorylation) and inhibition (by orthophosphate) were considerably less pronounced at 40 °C than at 30 °C. In general, the allosteric control of PhK's activity is definitely more subtle at body temperature. Changes in behavior related to activity at 40 °C and its control can be explained by the near disappearance of hysteresis at physiological temperature. In important ways, the picture of PhK that has emerged from six decades of study at temperatures of ≤30 °C does not coincide with that of the enzyme studied at physiological temperature. The probable underlying mechanism for the dramatic increase in PhK's activity between 30 and 40 °C is an abrupt change in the conformations of the regulatory β and catalytic γ subunits between these two temperatures.

  15. Space Experiment on Tuber Development and Starch Accumulation for CELSS

    NASA Technical Reports Server (NTRS)

    Tibbitts,Theodore W.; Croxdale, Judith C.; Brown, Christopher S.

    1997-01-01

    Potato explants (leaf, small stem section, and axillary bud), flown on STS-73, developed tubers of 1.5 cm diameter and 1.7 g mass during the 16 day period of spaceflight. The experiment was undertaken in the ASTROCULTURE(Trademark) experiment package under controlled temperature, humidity, lighting, and carbon dioxide concentrations. The tubers formed in the explant system under microgravity had the same gross morphology, the same anatomical configuration of cells and tissues, and the same sizes, shapes, and surface character of starch granules as tubers formed in a 1 g environment. The total accumulation of starch and other energy containing compounds was singular in space flight and ground control tubers. Enzyme activity of starch synthase, starch phosphorylase, and total hydrolase was similar in spaceflight and ground controls but activity of ADP-glucose pyrophosphorylase was reduced in the spaceflight tuber tissue. This experiment documented that potatoes will metabolize and accumulate starch as effectively in spaceflight as on the ground and thus this data provides the potential for effective utilization of potatoes in life support systems of space bases.

  16. Experiment 9: ASTROCULTURE: Growth and Starch Accumulation of Potato Tuber

    NASA Technical Reports Server (NTRS)

    Tibbitts, Theodore W.; Brown, Christopher S.; Croxdale, Judith G.; Wheeler, Raymond M.

    1998-01-01

    Potato explants (leaf, small stem section, and axillary bud) flown on STS-73 developed tubers of 1.5 cm diameter and 1.7 g mass during the 16-day period of space flight. The experiment was undertaken in the ASTROCULTURE(TM) experiment package under controlled temperature, humidity, lighting, and carbon dioxide concentrations. The tubers that formed in the explant system under microgravity had the same gross morphology, the same anatomical configuration of cells and tissues, and the same sizes, shapes, and surface character of starch granules as tubers formed in a 1 g environment. The total accumulation of starch and other energy containing compounds was similar in space flight and ground control tubers. Enzyme activity of starch synthase, starch phosphorylase, and total hydrolase was similar in space flight and ground controls, but activity of ADP-glucose pyrophosphorylase was reduced in the space flight tuber tissue. This experiment documented that potatoes will metabolize and accumulate starch as effectively in space flight as on the ground. Thus, this data provides the potential for effective utilization of potatoes in life support systems of space bases.

  17. New Starch Phenotypes Produced by TILLING in Barley

    PubMed Central

    Sparla, Francesca; Falini, Giuseppe; Botticella, Ermelinda; Pirone, Claudia; Talamè, Valentina; Bovina, Riccardo; Salvi, Silvio; Tuberosa, Roberto; Sestili, Francesco; Trost, Paolo

    2014-01-01

    Barley grain starch is formed by amylose and amylopectin in a 1∶3 ratio, and is packed into granules of different dimensions. The distribution of granule dimension is bimodal, with a majority of small spherical B-granules and a smaller amount of large discoidal A-granules containing the majority of the starch. Starch granules are semi-crystalline structures with characteristic X-ray diffraction patterns. Distinct features of starch granules are controlled by different enzymes and are relevant for nutritional value or industrial applications. Here, the Targeting-Induced Local Lesions IN Genomes (TILLING) approach was applied on the barley TILLMore TILLING population to identify 29 new alleles in five genes related to starch metabolism known to be expressed in the endosperm during grain filling: BMY1 (Beta-amylase 1), GBSSI (Granule Bound Starch Synthase I), LDA1 (Limit Dextrinase 1), SSI (Starch Synthase I), SSIIa (Starch Synthase IIa). Reserve starch of nine M3 mutant lines carrying missense or nonsense mutations was analysed for granule size, crystallinity and amylose/amylopectin content. Seven mutant lines presented starches with different features in respect to the wild-type: (i) a mutant line with a missense mutation in GBSSI showed a 4-fold reduced amylose/amylopectin ratio; (ii) a missense mutations in SSI resulted in 2-fold increase in A:B granule ratio; (iii) a nonsense mutation in SSIIa was associated with shrunken seeds with a 2-fold increased amylose/amylopectin ratio and different type of crystal packing in the granule; (iv) the remaining four missense mutations suggested a role of LDA1 in granule initiation, and of SSIIa in determining the size of A-granules. We demonstrate the feasibility of the TILLING approach to identify new alleles in genes related to starch metabolism in barley. Based on their novel physicochemical properties, some of the identified new mutations may have nutritional and/or industrial applications. PMID:25271438

  18. Stimulating effect of phosphatidic acid on autophosphorylation of phosphorylase kinase.

    PubMed

    Negami, A I; Sasaki, H; Yamamura, H

    1985-09-16

    Autophosphorylation of phosphorylase kinase from rabbit skeletal muscle was stimulated by acidic phospholipids such as phosphatidic acid (PA), phosphatidylinositol, and phosphatidyl-serine. PA stimulated an initial velocity of autophosphorylation 3.8-fold. When fully autophosphorylated, about 11 mol of phosphate per tetramer (alpha beta gamma delta) were incorporated in the presence of PA and about 6.5 mol in the absence of PA. In the presence of PA (100 micrograms/ml), there was a concomitant enhancement of its kinase activity about 25-fold at pH 6.8. PA (100 micrograms/ml) sharply decreased an apparent Ka for Ca2+ on autophosphorylation from 4.0 X 10(-5) M to 1.0 X 10(-6) M. Available evidence indicates that the Ca2+-activated, PA-dependent autophosphorylation of phosphorylase kinase shows an ability to stimulate glycogen breakdown.

  19. Two carbon fluxes to reserve starch in potato (Solanum tuberosum L.) tuber cells are closely interconnected but differently modulated by temperature.

    PubMed

    Fettke, Joerg; Leifels, Lydia; Brust, Henrike; Herbst, Karoline; Steup, Martin

    2012-05-01

    Parenchyma cells from tubers of Solanum tuberosum L. convert several externally supplied sugars to starch but the rates vary largely. Conversion of glucose 1-phosphate to starch is exceptionally efficient. In this communication, tuber slices were incubated with either of four solutions containing equimolar [U-¹⁴C]glucose 1-phosphate, [U-¹⁴C]sucrose, [U-¹⁴C]glucose 1-phosphate plus unlabelled equimolar sucrose or [U-¹⁴C]sucrose plus unlabelled equimolar glucose 1-phosphate. C¹⁴-incorporation into starch was monitored. In slices from freshly harvested tubers each unlabelled compound strongly enhanced ¹⁴C incorporation into starch indicating closely interacting paths of starch biosynthesis. However, enhancement disappeared when the tubers were stored. The two paths (and, consequently, the mutual enhancement effect) differ in temperature dependence. At lower temperatures, the glucose 1-phosphate-dependent path is functional, reaching maximal activity at approximately 20 °C but the flux of the sucrose-dependent route strongly increases above 20 °C. Results are confirmed by in vitro experiments using [U-¹⁴C]glucose 1-phosphate or adenosine-[U-¹⁴C]glucose and by quantitative zymograms of starch synthase or phosphorylase activity. In mutants almost completely lacking the plastidial phosphorylase isozyme(s), the glucose 1-phosphate-dependent path is largely impeded. Irrespective of the size of the granules, glucose 1-phosphate-dependent incorporation per granule surface area is essentially equal. Furthermore, within the granules no preference of distinct glucosyl acceptor sites was detectable. Thus, the path is integrated into the entire granule biosynthesis. In vitro C¹⁴C-incorporation into starch granules mediated by the recombinant plastidial phosphorylase isozyme clearly differed from the in situ results. Taken together, the data clearly demonstrate that two closely but flexibly interacting general paths of starch biosynthesis are functional

  20. Polynucleotide phosphorylase negatively controls spv virulence gene expression in Salmonella enterica.

    PubMed

    Ygberg, Sofia Eriksson; Clements, Mark O; Rytkönen, Anne; Thompson, Arthur; Holden, David W; Hinton, Jay C D; Rhen, Mikael

    2006-02-01

    Mutational inactivation of the cold-shock-associated exoribonuclease polynucleotide phosphorylase (PNPase; encoded by the pnp gene) in Salmonella enterica serovar Typhimurium was previously shown to enable the bacteria to cause chronic infection and to affect the bacterial replication in BALB/c mice (M. O. Clements et al., Proc. Natl. Acad. Sci. USA 99:8784-8789, 2002). Here, we report that PNPase deficiency results in increased expression of Salmonella plasmid virulence (spv) genes under in vitro growth conditions that allow induction of spv expression. Furthermore, whole-genome microarray-based transcriptome analyses of bacteria growing inside murine macrophage-like J774.A.1 cells revealed six genes as being significantly up-regulated in the PNPase-deficient background, which included spvABC, rtcB, entC, and STM2236. Mutational inactivation of the spvR regulator diminished the increased expression of spv observed in the pnp mutant background, implying that PNPase acts upstream of or at the level of SpvR. Finally, competition experiments revealed that the growth advantage of the pnp mutant in BALB/c mice was dependent on spvR as well. Combined, our results support the idea that in S. enterica PNPase, apart from being a regulator of the cold shock response, also functions in tuning the expression of virulence genes and bacterial fitness during infection. PMID:16428774

  1. Thermal properties of barley starch and its relation to starch characteristics.

    PubMed

    Källman, Anna; Vamadevan, Varatharajan; Bertoft, Eric; Koch, Kristine; Seetharaman, Koushik; Åman, Per; Andersson, Roger

    2015-11-01

    Amylopectin fine structure and starch gelatinization and retrogradation were studied in 10 different barley cultivars/breeding lines. Clusters and building blocks were isolated from the amylopectin by α-amylase from Bacillus amyloliquefaciens and their structure was characterized. Gelatinization was studied at a starch:water ratio of 1:3, and retrogradation was studied on gelatinized starch at starch:water ratio of 1:2, by differential scanning calorimetry. Three barley cultivars/breeding lines possessed the amo1 mutation, and they all had a lower molar proportion of chains of DP ≥38 and more of large building blocks. The amo1 mutation also resulted in a higher gelatinization temperature and a broader temperature interval during gelatinization. Overall, small clusters with a dense structure resulted in a higher gelatinization temperature while retrogradation was promoted by short chains in the amylopectin and many large building blocks.

  2. Degradation of Glucan Primers in the Absence of Starch Synthase 4 Disrupts Starch Granule Initiation in Arabidopsis.

    PubMed

    Seung, David; Lu, Kuan-Jen; Stettler, Michaela; Streb, Sebastian; Zeeman, Samuel C

    2016-09-23

    Arabidopsis leaf chloroplasts typically contain five to seven semicrystalline starch granules. It is not understood how the synthesis of each granule is initiated or how starch granule number is determined within each chloroplast. An Arabidopsis mutant lacking the glucosyl-transferase, STARCH SYNTHASE 4 (SS4) is impaired in its ability to initiate starch granules; its chloroplasts rarely contain more than one large granule, and the plants have a pale appearance and reduced growth. Here we report that the chloroplastic α-amylase AMY3, a starch-degrading enzyme, interferes with granule initiation in the ss4 mutant background. The amy3 single mutant is similar in phenotype to the wild type under normal growth conditions, with comparable numbers of starch granules per chloroplast. Interestingly, the ss4 mutant displays a pleiotropic reduction in the activity of AMY3. Remarkably, complete abolition of AMY3 (in the amy3 ss4 double mutant) increases the number of starch granules produced in each chloroplast, suppresses the pale phenotype of ss4, and nearly restores normal growth. The amy3 mutation also restores starch synthesis in the ss3 ss4 double mutant, which lacks STARCH SYNTHASE 3 (SS3) in addition to SS4. The ss3 ss4 line is unable to initiate any starch granules and is thus starchless. We suggest that SS4 plays a key role in granule initiation, allowing it to proceed in a way that avoids premature degradation of primers by starch hydrolases, such as AMY3. PMID:27458017

  3. Degradation of Glucan Primers in the Absence of Starch Synthase 4 Disrupts Starch Granule Initiation in Arabidopsis*

    PubMed Central

    Lu, Kuan-Jen; Stettler, Michaela; Streb, Sebastian

    2016-01-01

    Arabidopsis leaf chloroplasts typically contain five to seven semicrystalline starch granules. It is not understood how the synthesis of each granule is initiated or how starch granule number is determined within each chloroplast. An Arabidopsis mutant lacking the glucosyl-transferase, STARCH SYNTHASE 4 (SS4) is impaired in its ability to initiate starch granules; its chloroplasts rarely contain more than one large granule, and the plants have a pale appearance and reduced growth. Here we report that the chloroplastic α-amylase AMY3, a starch-degrading enzyme, interferes with granule initiation in the ss4 mutant background. The amy3 single mutant is similar in phenotype to the wild type under normal growth conditions, with comparable numbers of starch granules per chloroplast. Interestingly, the ss4 mutant displays a pleiotropic reduction in the activity of AMY3. Remarkably, complete abolition of AMY3 (in the amy3 ss4 double mutant) increases the number of starch granules produced in each chloroplast, suppresses the pale phenotype of ss4, and nearly restores normal growth. The amy3 mutation also restores starch synthesis in the ss3 ss4 double mutant, which lacks STARCH SYNTHASE 3 (SS3) in addition to SS4. The ss3 ss4 line is unable to initiate any starch granules and is thus starchless. We suggest that SS4 plays a key role in granule initiation, allowing it to proceed in a way that avoids premature degradation of primers by starch hydrolases, such as AMY3. PMID:27458017

  4. Computer-generated Model of Purine Nucleoside Phosphorylase (PNP)

    NASA Technical Reports Server (NTRS)

    1987-01-01

    Purine Nucleoside Phosphorylase (PNP) is an important target enzyme for the design of anti-cancer and immunosuppressive drugs. Bacterial PNP, which is slightly different from the human enzyme, is used to synthesize chemotherapuautic agents. Knowledge of the three-dimensional structure of the bacterial PNP molecule is useful in efforts to engineer different types of PNP enzymes, that can be used to produce new chemotherapeutic agents. This picture shows a computer model of bacterial PNP, which looks a lot like a display of colorful ribbons. Principal Investigator was Charles Bugg.

  5. Substrate specificity of pyrimidine nucleoside phosphorylases of NP-II family probed by X-ray crystallography and molecular modeling

    NASA Astrophysics Data System (ADS)

    Balaev, V. V.; Lashkov, A. A.; Prokofev, I. I.; Gabdulkhakov, A. G.; Seregina, T. A.; Mironov, A. S.; Betzel, C.; Mikhailov, A. M.

    2016-09-01

    Pyrimidine nucleoside phosphorylases, which are widely used in the biotechnological production of nucleosides, have different substrate specificity for pyrimidine nucleosides. An interesting feature of these enzymes is that the three-dimensional structure of thymidine-specific nucleoside phosphorylase is similar to the structure of nonspecific pyrimidine nucleoside phosphorylase. The three-dimensional structures of thymidine phosphorylase from Salmonella typhimurium and nonspecific pyrimidine nucleoside phosphorylase from Bacillus subtilis in complexes with a sulfate anion were determined for the first time by X-ray crystallography. An analysis of the structural differences between these enzymes demonstrated that Lys108, which is involved in the phosphate binding in pyrimidine nucleoside phosphorylase, corresponds to Met111 in thymidine phosphorylases. This difference results in a decrease in the charge on one of the hydroxyl oxygens of the phosphate anion in thymidine phosphorylase and facilitates the catalysis through SN2 nucleophilic substitution. Based on the results of X-ray crystallography, the virtual screening was performed for identifying a potent inhibitor (anticancer agent) of nonspecific pyrimidine nucleoside phosphorylase, which does not bind to thymidine phosphorylase. The molecular dynamics simulation revealed the stable binding of the discovered compound—2-pyrimidin-2-yl-1H-imidazole-4-carboxylic acid—to the active site of pyrimidine nucleoside phosphorylase.

  6. Extruding foams from corn starch acetate and native corn starch.

    PubMed

    Guan, Junjie; Hanna, Milford A

    2004-01-01

    Because of the hydrophilic characteristics of native starch foams and the cost of modifying starch, the uses of starch and modified starch foams are hindered. To decrease hydrophilicity and cost of starch foams, native corn starch was blended with starch acetate and extruded. A twin-screw mixing extruder was used to produce the foams. Native starch content, screw speed, and barrel temperature had significant effects on molecular degradation of starches during extrusion. The melting temperature of extruded starch acetate/native starch foam was higher (216 degrees C) than that for starch acetate (193.4 degrees C). Strong peaks in the X-ray diffractograms of extruded starch acetate/native starch foam suggested new crystalline regions were formed. Optimum conditions for high radial expansion ratio, high compressibility, low specific mechanical energy requirement, and low water absorption index were 46.0% native starch content, 163 rpm screw speed, and 148 degrees C barrel temperature.

  7. Structural bases for N-glycan processing by mannoside phosphorylase.

    PubMed

    Ladevèze, Simon; Cioci, Gianluca; Roblin, Pierre; Mourey, Lionel; Tranier, Samuel; Potocki-Véronèse, Gabrielle

    2015-06-01

    The first crystal structure of Uhgb_MP, a β-1,4-mannopyranosyl-chitobiose phosphorylase belonging to the GH130 family which is involved in N-glycan degradation by human gut bacteria, was solved at 1.85 Å resolution in the apo form and in complex with mannose and N-acetylglucosamine. SAXS and crystal structure analysis revealed a hexameric structure, a specific feature of GH130 enzymes among other glycoside phosphorylases. Mapping of the -1 and +1 subsites in the presence of phosphate confirmed the conserved Asp104 as the general acid/base catalytic residue, which is in agreement with a single-step reaction mechanism involving Man O3 assistance for proton transfer. Analysis of this structure, the first to be solved for a member of the GH130_2 subfamily, revealed Met67, Phe203 and the Gly121-Pro125 loop as the main determinants of the specificity of Uhgb_MP and its homologues towards the N-glycan core oligosaccharides and mannan, and the molecular bases of the key role played by GH130 enzymes in the catabolism of dietary fibre and host glycans.

  8. Structural bases for N-glycan processing by mannoside phosphorylase

    PubMed Central

    Ladevèze, Simon; Cioci, Gianluca; Roblin, Pierre; Mourey, Lionel; Tranier, Samuel; Potocki-Véronèse, Gabrielle

    2015-01-01

    The first crystal structure of Uhgb_MP, a β-1,4-mannopyranosyl-chitobiose phosphorylase belonging to the GH130 family which is involved in N-glycan degradation by human gut bacteria, was solved at 1.85 Å resolution in the apo form and in complex with mannose and N-acetylglucosamine. SAXS and crystal structure analysis revealed a hexameric structure, a specific feature of GH130 enzymes among other glycoside phosphorylases. Mapping of the −1 and +1 subsites in the presence of phosphate confirmed the conserved Asp104 as the general acid/base catalytic residue, which is in agreement with a single-step reaction mechanism involving Man O3 assistance for proton transfer. Analysis of this structure, the first to be solved for a member of the GH130_2 subfamily, revealed Met67, Phe203 and the Gly121–Pro125 loop as the main determinants of the specificity of Uhgb_MP and its homologues towards the N-glycan core oligosaccharides and mannan, and the molecular bases of the key role played by GH130 enzymes in the catabolism of dietary fibre and host glycans. PMID:26057673

  9. Doubling Power Output of Starch Biobattery Treated by the Most Thermostable Isoamylase from an Archaeon Sulfolobus tokodaii

    PubMed Central

    Cheng, Kun; Zhang, Fei; Sun, Fangfang; Chen, Hongge; Percival Zhang, Y-H

    2015-01-01

    Biobattery, a kind of enzymatic fuel cells, can convert organic compounds (e.g., glucose, starch) to electricity in a closed system without moving parts. Inspired by natural starch metabolism catalyzed by starch phosphorylase, isoamylase is essential to debranch alpha-1,6-glycosidic bonds of starch, yielding linear amylodextrin – the best fuel for sugar-powered biobattery. However, there is no thermostable isoamylase stable enough for simultaneous starch gelatinization and enzymatic hydrolysis, different from the case of thermostable alpha-amylase. A putative isoamylase gene was mined from megagenomic database. The open reading frame ST0928 from a hyperthermophilic archaeron Sulfolobus tokodaii was cloned and expressed in E. coli. The recombinant protein was easily purified by heat precipitation at 80 oC for 30 min. This enzyme was characterized and required Mg2+ as an activator. This enzyme was the most stable isoamylase reported with a half lifetime of 200 min at 90 oC in the presence of 0.5 mM MgCl2, suitable for simultaneous starch gelatinization and isoamylase hydrolysis. The cuvett-based air-breathing biobattery powered by isoamylase-treated starch exhibited nearly doubled power outputs than that powered by the same concentration starch solution, suggesting more glucose 1-phosphate generated. PMID:26289411

  10. Doubling Power Output of Starch Biobattery Treated by the Most Thermostable Isoamylase from an Archaeon Sulfolobus tokodaii.

    PubMed

    Cheng, Kun; Zhang, Fei; Sun, Fangfang; Chen, Hongge; Percival Zhang, Y-H

    2015-08-20

    Biobattery, a kind of enzymatic fuel cells, can convert organic compounds (e.g., glucose, starch) to electricity in a closed system without moving parts. Inspired by natural starch metabolism catalyzed by starch phosphorylase, isoamylase is essential to debranch alpha-1,6-glycosidic bonds of starch, yielding linear amylodextrin - the best fuel for sugar-powered biobattery. However, there is no thermostable isoamylase stable enough for simultaneous starch gelatinization and enzymatic hydrolysis, different from the case of thermostable alpha-amylase. A putative isoamylase gene was mined from megagenomic database. The open reading frame ST0928 from a hyperthermophilic archaeron Sulfolobus tokodaii was cloned and expressed in E. coli. The recombinant protein was easily purified by heat precipitation at 80 (o)C for 30 min. This enzyme was characterized and required Mg(2+) as an activator. This enzyme was the most stable isoamylase reported with a half lifetime of 200 min at 90 (o)C in the presence of 0.5 mM MgCl2, suitable for simultaneous starch gelatinization and isoamylase hydrolysis. The cuvett-based air-breathing biobattery powered by isoamylase-treated starch exhibited nearly doubled power outputs than that powered by the same concentration starch solution, suggesting more glucose 1-phosphate generated.

  11. Unusual starch degradation pathway via cyclodextrins in the hyperthermophilic sulfate-reducing archaeon Archaeoglobus fulgidus strain 7324.

    PubMed

    Labes, Antje; Schönheit, Peter

    2007-12-01

    The hyperthermophilic archaeon Archaeoglobus fulgidus strain 7324 has been shown to grow on starch and sulfate and thus represents the first sulfate reducer able to degrade polymeric sugars. The enzymes involved in starch degradation to glucose 6-phosphate were studied. In extracts of starch-grown cells the activities of the classical starch degradation enzymes, alpha-amylase and amylopullulanase, could not be detected. Instead, evidence is presented here that A. fulgidus utilizes an unusual pathway of starch degradation involving cyclodextrins as intermediates. The pathway comprises the combined action of an extracellular cyclodextrin glucanotransferase (CGTase) converting starch to cyclodextrins and the intracellular conversion of cyclodextrins to glucose 6-phosphate via cyclodextrinase (CDase), maltodextrin phosphorylase (Mal-P), and phosphoglucomutase (PGM). These enzymes, which are all induced after growth on starch, were characterized. CGTase catalyzed the conversion of starch to mainly beta-cyclodextrin. The gene encoding CGTase was cloned and sequenced and showed highest similarity to a glucanotransferase from Thermococcus litoralis. After transport of the cyclodextrins into the cell by a transport system to be defined, these molecules are linearized via a CDase, catalyzing exclusively the ring opening of the cyclodextrins to the respective maltooligodextrins. These are degraded by a Mal-P to glucose 1-phosphate. Finally, PGM catalyzes the conversion of glucose 1-phosphate to glucose 6-phosphate, which is further degraded to pyruvate via the modified Embden-Meyerhof pathway.

  12. A conserved loop in polynucleotide phosphorylase (PNPase) essential for both RNA and ADP/phosphate binding.

    PubMed

    Carzaniga, Thomas; Mazzantini, Elisa; Nardini, Marco; Regonesi, Maria Elena; Greco, Claudio; Briani, Federica; De Gioia, Luca; Dehò, Gianni; Tortora, Paolo

    2014-02-01

    Polynucleotide phosphorylase (PNPase) reversibly catalyzes RNA phosphorolysis and polymerization of nucleoside diphosphates. Its homotrimeric structure forms a central channel where RNA is accommodated. Each protomer core is formed by two paralogous RNase PH domains: PNPase1, whose function is largely unknown, hosts a conserved FFRR loop interacting with RNA, whereas PNPase2 bears the putative catalytic site, ∼20 Å away from the FFRR loop. To date, little is known regarding PNPase catalytic mechanism. We analyzed the kinetic properties of two Escherichia coli PNPase mutants in the FFRR loop (R79A and R80A), which exhibited a dramatic increase in Km for ADP/Pi binding, but not for poly(A), suggesting that the two residues may be essential for binding ADP and Pi. However, both mutants were severely impaired in shifting RNA electrophoretic mobility, implying that the two arginines contribute also to RNA binding. Additional interactions between RNA and other PNPase domains (such as KH and S1) may preserve the enzymatic activity in R79A and R80A mutants. Inspection of enzyme structure showed that PNPase has evolved a long-range acting hydrogen bonding network that connects the FFRR loop with the catalytic site via the F380 residue. This hypothesis was supported by mutation analysis. Phylogenetic analysis of PNPase domains and RNase PH suggests that such network is a unique feature of PNPase1 domain, which coevolved with the paralogous PNPase2 domain.

  13. Nucleoside Diphosphate Sugar-Starch Glucosyl Transferase Activity of wx Starch Granules 1

    PubMed Central

    Nelson, Oliver E.; Chourey, Prem S.; Chang, Ming Tu

    1978-01-01

    Starch granule preparations from the endosperm tissue of all waxy maize (Zea mays L.) mutants tested have low and approximately equal capability to incorporate glucose from adenosine diphosphate glucose into starch. As the substrate concentration is reduced, however, the activity of waxy preparations relative to nonmutant increases until, at the lowest substrate concentration utilized (0.1 μM), the activity of the waxy preparations is nearly equal to that of the nonmutant preparation. The apparent Km (adenosine diphosphate glucose) for starch granule preparations from wx-C/wx-C/wx-C endosperms was 7.1 × 10−5 M, which is compared to 3 × 10−3 M for preparations from nonwaxy endosperms. Starch granule preparations from three other waxy mutants of independent mutational origin have levels of enzymic activity approximately equal to wx-C at a given substrate concentration giving rise to similar apparent Km estimates. We conclude that there is in maize endosperm starch granules a second starch granule-bound glycosyl transferase, whose presence is revealed when mutation eliminates activity of the more active glucosyl transferase catalyzing the same reaction. PMID:16660522

  14. Branching patterns in leaf starches from Arabidopsis mutants deficient in diverse starch synthases.

    PubMed

    Zhu, Fan; Bertoft, Eric; Szydlowski, Nicolas; d'Hulst, Christophe; Seetharaman, Koushik

    2015-01-12

    This is the first report on the cluster structure of transitory starch from Arabidopsis leaves. In addition to wild type, the molecular structures of leaf starch from mutants deficient in starch synthases (SS) including single enzyme mutants ss1-, ss2-, or ss3-, and also double mutants ss1-ss2- and ss1-ss3- were characterized. The mutations resulted in increased amylose content. Clusters from whole starch were isolated by partial hydrolysis using α-amylase of Bacillus amyloliquefaciens. The clusters were then further hydrolyzed with concentrated α-amylase of B. amyloliquefaciens to produce building blocks (α-limit dextrins). Structures of the clusters and their building blocks were characterized by chromatography of samples before and after debranching treatment. While the mutations increased the size of clusters, the reasons were different as reflected by the composition of their unit chains and building blocks. In general, all mutants contained more of a-chains that preferentially increased the number of small building blocks with only two chains. The clusters of the double mutant ss1-ss3- were very large and possessed also more of large building blocks with four or more chains. The results from transitory starch are compared with those from agriculturally important crops in the context that to what extent the Arabidopsis can be a true biotechnological reflection for starch modifications through genetic means.

  15. Starch structure in developing barley endosperm.

    PubMed

    Källman, Anna; Bertoft, Eric; Koch, Kristine; Sun, Chuanxin; Åman, Per; Andersson, Roger

    2015-11-01

    Barley spikes of the cultivars/breeding lines Gustav, Karmosé and SLU 7 were harvested at 9, 12 and 24 days after flowering in order to study starch structure in developing barley endosperm. Kernel dry weight, starch content and amylose content increased during development. Structural analysis was performed on whole starch and included the chain-length distribution of the whole starches and their β-limit dextrins. Karmosé, possessing the amo1 mutation, had higher amylose content and a lower proportion of long chains (DP ≥38) in the amylopectin component than SLU 7 and Gustav. Structural differences during endosperm development were seen as a decrease in molar proportion of chains of DP 22-37 in whole starch. In β-limit dextrins, the proportion of Bfp-chains (DP 4-7) increased and the proportion of BSmajor-chains (DP 15-27) decreased during development, suggesting more frequent activity of starch branching enzymes at later stages of maturation, resulting in amylopectin with denser structure.

  16. Polynucleotide phosphorylase has an impact on cell biology of Campylobacter jejuni

    PubMed Central

    Haddad, Nabila; Tresse, Odile; Rivoal, Katell; Chevret, Didier; Nonglaton, Quentin; Burns, Christopher M.; Prévost, Hervé; Cappelier, Jean M.

    2012-01-01

    Polynucleotide phosphorylase (PNPase), encoded by the pnp gene, is known to degrade mRNA, mediating post-transcriptional regulation and may affect cellular functions. The role of PNPase is pleiotropic. As orthologs of the two major ribonucleases (RNase E and RNase II) of Escherichia coli are missing in the Campylobacter jejuni genome, in the current study the focus has been on the C. jejuni ortholog of PNPase. The effect of PNPase mutation on C. jejuni phenotypes and proteome was investigated. The inactivation of the pnp gene reduced significantly the ability of C. jejuni to adhere and to invade Ht-29 cells. Moreover, the pnp mutant strain exhibited a decrease in C. jejuni swimming ability and chick colonization. To explain effects of PNPase on C. jejuni 81-176 phenotype, the proteome of the pnp mutant and parental strains were compared. Overall, little variation in protein production was observed. Despite the predicted role of PNPase in mRNA regulation, the pnp mutation did not induce profound proteomic changes suggesting that other ribonucleases in C. jejuni might ensure this biological function in the absence of PNPase. Nevertheless, synthesis of proteins which are involved in virulence (LuxS, PEB3), motility (N-acetylneuraminic acid synthetase), stress-response (KatA, DnaK, Hsp90), and translation system (EF-Tu, EF-G) were modified in the pnp mutant strain suggesting a more specific role of PNPase in C. jejuni. In conclusion, PNPase deficiency induces limited but important consequences on C. jejuni biology that could explain swimming limitation, chick colonization delay, and the decrease of cell adhesion/invasion ability. PMID:22919622

  17. Bacillus subtilis polynucleotide phosphorylase 3'-to-5' DNase activity is involved in DNA repair.

    PubMed

    Cardenas, Paula P; Carrasco, Begoña; Sanchez, Humberto; Deikus, Gintaras; Bechhofer, David H; Alonso, Juan C

    2009-07-01

    In the presence of Mn(2+), an activity in a preparation of purified Bacillus subtilis RecN degrades single-stranded (ss) DNA with a 3' --> 5' polarity. This activity is not associated with RecN itself, because RecN purified from cells lacking polynucleotide phosphorylase (PNPase) does not show the exonuclease activity. We show here that, in the presence of Mn(2+) and low-level inorganic phosphate (P(i)), PNPase degrades ssDNA. The limited end-processing of DNA is regulated by ATP and is inactive in the presence of Mg(2+) or high-level P(i). In contrast, the RNase activity of PNPase requires Mg(2+) and P(i), suggesting that PNPase degradation of RNA and ssDNA occur by mutually exclusive mechanisms. A null pnpA mutation (DeltapnpA) is not epistatic with Delta recA, but is epistatic with DeltarecN and Delta ku, which by themselves are non-epistatic. The addA5, Delta recO, Delta recQ (Delta recJ), Delta recU and Delta recG mutations (representative of different epistatic groups), in the context of DeltapnpA, demonstrate gain- or loss-of-function by inactivation of repair-by-recombination, depending on acute or chronic exposure to the damaging agent and the nature of the DNA lesion. Our data suggest that PNPase is involved in various nucleic acid metabolic pathways, and its limited ssDNA exonuclease activity plays an important role in RecA-dependent and RecA-independent repair pathways. PMID:19433509

  18. Further characterization of the polynucleotide phosphorylase of Micrococcus luteus

    PubMed Central

    Letendre, Carol H.; Singer, Maxine F.

    1975-01-01

    The purification of polynucleotide phosphorylase from Micrococcus luteus by chromatography on phosphocellulose columns is described. This procedure offers several advantages over previous procedures. Previously determined molecular weights for Form-I enzyme and Form-T enzyme derived from Form-I by limited tryptic hydrolysis were confirmed as 2.7 and 2.3 × 105, respectively. Form-I appears homogeneous in the ultracentrifuge, but multiple active protein species are separable by polyacrylamide gel electrophoresis. The multiple species are probably the result of proteolysis. On polyacrylamide gel electrophoresis under denaturing conditions, Form-T yielded a single size of subunit of 71,000 daltons, and Form-I yielded several bands of different molecular sizes. These results differ from earlier determinations. The amino acid compositions of Form-I and Form-T are reported. Form-I contains only between 8 and 10 cysteine residues per molecule and Form-T half that many. Images PMID:1121422

  19. Molecular mechanisms of McArdle's disease (muscle glycogen phosphorylase deficiency). RNA and DNA analysis.

    PubMed Central

    Gautron, S; Daegelen, D; Mennecier, F; Dubocq, D; Kahn, A; Dreyfus, J C

    1987-01-01

    Lack of muscle glycogen phosphorylase activity leads to McArdle's disease, a rare metabolic myopathy. To investigate its molecular basis at the nucleic acid level, we isolated muscle phosphorylase cDNA clones from a human cDNA library in Escherichia coli plasmid pBR 322. Subcloning of one insertion of M13 bacteriophage permitted its definite identification by sequencing. Northern blot experiments revealed one specific messenger RNA of 3.4 kilobases found uniquely in tissues expressing muscle phosphorylase. We show that McArdle's disease exhibits a molecular heterogeneity at the messenger RNA level. In eight unrelated cases of McArdle's disease in which no inactive proteins had been detected, we assayed muscle biopsies for phosphorylase mRNA by Northern blotting. In five cases, no muscle phosphorylase mRNA could be detected, while in three other cases, normal length mRNA was present in lower amounts. Moreover, Southern blot analysis of DNA isolated from white blood cells in four McArdle patients revealed no major deletion or rearrangements of the phosphorylase gene as compared with controls. Images PMID:3466902

  20. Computer Simulations Reveal Substrate Specificity of Glycosidic Bond Cleavage in Native and Mutant Human Purine Nucleoside Phosphorylase.

    PubMed

    Isaksen, Geir Villy; Hopmann, Kathrin Helen; Åqvist, Johan; Brandsdal, Bjørn Olav

    2016-04-12

    Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of purine ribonucleosides and 2'-deoxyribonucleosides, yielding the purine base and (2'-deoxy)ribose 1-phosphate as products. While this enzyme has been extensively studied, several questions with respect to the catalytic mechanism have remained largely unanswered. The role of the phosphate and key amino acid residues in the catalytic reaction as well as the purine ring protonation state is elucidated using density functional theory calculations and extensive empirical valence bond (EVB) simulations. Free energy surfaces for adenosine, inosine, and guanosine are fitted to ab initio data and yield quantitative agreement with experimental data when the surfaces are used to model the corresponding enzymatic reactions. The cognate substrates 6-aminopurines (inosine and guanosine) interact with PNP through extensive hydrogen bonding, but the substrate specificity is found to be a direct result of the electrostatic preorganization energy along the reaction coordinate. Asn243 has previously been identified as a key residue providing substrate specificity. Mutation of Asn243 to Asp has dramatic effects on the substrate specificity, making 6-amino- and 6-oxopurines equally good as substrates. The principal effect of this particular mutation is the change in the electrostatic preorganization energy between the native enzyme and the Asn243Asp mutant, clearly favoring adenosine over inosine and guanosine. Thus, the EVB simulations show that this particular mutation affects the electrostatic preorganization of the active site, which in turn can explain the substrate specificity. PMID:26985580

  1. Effects of Bay m 1099, an α-Glucosidase Inhibitor, on Starch Degradation in Germinating Mung Beans.

    PubMed

    Konishi, Y; Aitani, M; Nakatani, N

    1998-01-01

    To examine the mechanism of starch degradation in legume cotyledons and the physiological role of α-glucosidase, mung bean seeds were germinated in the presence of Bay m 1099, an α-glucosidase inhibitor. Bay m 1099 (10 μg/ml medium), which minimized the growth deterioration of the mung bean seedlings, caused no changes in the overall rate of starch degradation and of soluble carbohydrate production in the cotyledons, although α-glucosidase activity had been completely suppressed. Total amylase and phosphorylase activities were not influenced by Bay m 1099. These results suggest that the mung bean α-glucosidase is less responsible for starch degradation, unlike wheat α-glucosidase [Konishi et al., Biosci. Biotech. Biochem., 58, 135-139 (1994)].

  2. Crystal Structure and Substrate Recognition of Cellobionic Acid Phosphorylase, Which Plays a Key Role in Oxidative Cellulose Degradation by Microbes*

    PubMed Central

    Nam, Young-Woo; Nihira, Takanori; Arakawa, Takatoshi; Saito, Yuka; Kitaoka, Motomitsu; Nakai, Hiroyuki; Fushinobu, Shinya

    2015-01-01

    The microbial oxidative cellulose degradation system is attracting significant research attention after the recent discovery of lytic polysaccharide mono-oxygenases. A primary product of the oxidative and hydrolytic cellulose degradation system is cellobionic acid (CbA), the aldonic acid form of cellobiose. We previously demonstrated that the intracellular enzyme belonging to glycoside hydrolase family 94 from cellulolytic fungus and bacterium is cellobionic acid phosphorylase (CBAP), which catalyzes reversible phosphorolysis of CbA into glucose 1-phosphate and gluconic acid (GlcA). In this report, we describe the biochemical characterization and the three-dimensional structure of CBAP from the marine cellulolytic bacterium Saccharophagus degradans. Structures of ligand-free and complex forms with CbA, GlcA, and a synthetic disaccharide product from glucuronic acid were determined at resolutions of up to 1.6 Å. The active site is located near the dimer interface. At subsite +1, the carboxylate group of GlcA and CbA is recognized by Arg-609 and Lys-613. Additionally, one residue from the neighboring protomer (Gln-190) is involved in the carboxylate recognition of GlcA. A mutational analysis indicated that these residues are critical for the binding and catalysis of the aldonic and uronic acid acceptors GlcA and glucuronic acid. Structural and sequence comparisons with other glycoside hydrolase family 94 phosphorylases revealed that CBAPs have a unique subsite +1 with a distinct amino acid residue conservation pattern at this site. This study provides molecular insight into the energetically efficient metabolic pathway of oxidized sugars that links the oxidative cellulolytic pathway to the glycolytic and pentose phosphate pathways in cellulolytic microbes. PMID:26041776

  3. Starch conversion technology

    SciTech Connect

    Van Beynum, G.M.A.; Roels, J.A.

    1985-01-01

    This volume with contributions by 17 international experts provides an overview of processes by which starch is converted to a form which makes it more suitable for other applications. Products from starch biochemical conversions include organic acids, alcohol, bipolymers, enzymes, amino acids, antibiotics and hormones. Alcohol produced from starch can be used to reduce dependency on petroleum for energy. Literature references and a subject index are provided.

  4. Enzymic Mechanism of Starch Breakdown in Germinating Rice Seeds I. An Analytical Study1

    PubMed Central

    Murata, Takao; Akazawa, T.; Fukuchi, Shikiko

    1968-01-01

    Time-sequence analyses of carbohydrate breakdown in germinating rice seeds shows that a rapid breakdown of starch reserve in endosperm starts after about 4 days of germination. Although the major soluble carbohydrate in the dry seed is sucrose, a marked increase in the production of glucose and maltooligosaccharides accompanies the breakdown of starch. Maltotriose was found to constitute the greatest portion of the oligosaccharides throughout the germination stage. α-Amylase activities were found to parallel the pattern of starch breakdown. Assays for phosphorylase activity showed that this enzyme may account for much smaller amounts of starch breakdown per grain, as compared to the amounts hydrolyzed by α-amylase. There was a transient decline in the content of sucrose in the initial 4 days of seed germination, followed by the gradual increase in later germination stages. During the entire germination stage, sucrose synthetase activity was not detected in the endosperm, although appreciable enzyme activity was present in the growing shoot tissues as well as in the frozen rice seeds harvested at the mid-milky stage. We propose the predominant formation of glucose from starch reserves in the endosperm by the action of α-amylase and accompanying hydrolytic enzyme(s) and that this sugar is eventually mobilized to the growing tissues, shoots or roots. Images PMID:16656988

  5. Starch nanoparticles: a review.

    PubMed

    Le Corre, Déborah; Bras, Julien; Dufresne, Alain

    2010-05-10

    Starch is a natural, renewable, and biodegradable polymer produced by many plants as a source of stored energy. It is the second most abundant biomass material in nature. The starch structure has been under research for years, and because of its complexity, an universally accepted model is still lacking (Buleon, A.; et al. Int. J. Biol. Macromol. 1998, 23, 85-112). However, the predominant model for starch is a concentric semicrystalline multiscale structure that allows the production of new nanoelements: (i) starch nanocrystals resulting from the disruption of amorphous domains from semicrystalline granules by acid hydrolysis and (ii) starch nanoparticles produced from gelatinized starch. This paper intends to give a clear overview of starch nanoparticle preparation, characterization, properties, and applications. Recent studies have shown that they could be used as fillers to improve mechanical and barrier properties of biocomposites. Their use for industrial packaging, continuously looking for innovative solutions for efficient and sustainable systems, is being investigated. Therefore, recently, starch nanoparticles have been the focus of an exponentially increasing number of works devoted to develop biocomposites by blending starch nanoparticles with different biopolymeric matrices. To our knowledge, this topic has never been reviewed, despite several published strategies and conclusions. PMID:20405913

  6. Analysis of cytosolic heteroglycans from leaves of transgenic potato (Solanum tuberosum L.) plants that under- or overexpress the Pho 2 phosphorylase isozyme.

    PubMed

    Fettke, Joerg; Poeste, Simon; Eckermann, Nora; Tiessen, Axel; Pauly, Markus; Geigenberger, Peter; Steup, Martin

    2005-12-01

    During starch degradation, chloroplasts export neutral sugars into the cytosol where they appear to enter a complex glycan metabolism. Interactions between glycans and glucosyl transferases residing in the cytosol were studied by analyzing transgenic potato (Solanum tuberosum L.) plants that possess either decreased or elevated levels of the cytosolic (Pho 2) phosphorylase isoform. Water-soluble heteroglycans (SHGs) were isolated from these plants and were characterized. SHG contains, as major constituents, arabinose, rhamnose, galactose and glucose. Non-aqueous fractionation combined with other separation techniques revealed a distinct pool of the SHG that is located in the cytosol. Under in vitro conditions, the cytosolic heteroglycans act as glucosyl acceptor selectively for Pho 2. Acceptor sites were characterized by a specific hydrolytic degradation following the Pho 2-catalyzed glucosyl transfer. The size distribution of the cytosolic SHG increased during the dark period, indicating a distinct metabolic activity related to net starch degradation. Antisense inhibition of Pho 2 resulted in increased glucosyl and rhamnosyl contents of the glycans. Overexpression of Pho 2 decreased the content of both residues. Compared with the wild type, in both types of transgenic plants the size of the cytosolic glycans was increased.

  7. The crystal structure of Escherichia coli maltodextrin phosphorylase provides an explanation for the activity without control in this basic archetype of a phosphorylase.

    PubMed Central

    Watson, K A; Schinzel, R; Palm, D; Johnson, L N

    1997-01-01

    In animals, glycogen phosphorylase (GP) exists in an inactive (T state) and an active (R state) equilibrium that can be altered by allosteric effectors or covalent modification. In Escherichia coli, the activity of maltodextrin phosphorylase (MalP) is controlled by induction at the level of gene expression, and the enzyme exhibits no regulatory properties. We report the crystal structure of E. coli maltodextrin phosphorylase refined to 2.4 A resolution. The molecule consists of a dimer with 796 amino acids per monomer, with 46% sequence identity to the mammalian enzyme. The overall structure of MalP shows a similar fold to GP and the catalytic sites are highly conserved. However, the relative orientation of the two subunits in E. coli MalP is different from both the T and R state GP structures, and there are significant changes at the subunit-subunit interfaces. The sequence changes result in loss of each of the control sites present in rabbit muscle GP. As a result of the changes at the subunit interface, the 280s loop, which in T state GP acts as a gate to control access to the catalytic site, is held in an open conformation in MalP. The open access to the conserved catalytic site provides an explanation for the activity without control in this basic archetype of a phosphorylase. PMID:9009262

  8. Blue maize: morphology and starch synthase characterization of starch granule.

    PubMed

    Utrilla-Coello, Rubi G; Agama-Acevedo, Edith; de la Rosa, Ana Paulina Barba; Martinez-Salgado, Jose L; Rodriguez-Ambriz, Sandra L; Bello-Perez, Luis A

    2009-03-01

    The use of pigmented maize varieties has increased due to their high anthocyanins content, but very few studies are reported about the starch properties of these grains. The aim of this work was to isolate the starch granules from pigmented blue maize and carry out the morphological, physicochemical, and biochemical characterization studies. The proximate composition of starch granules showed high protein contents, after purification, the blue maize starch presented lower protein amount than starch from white maize (control). Although the purity of starch granules was increased, the damaged starch (determined for the Maltase cross absence) was also increased. Scanning electron microscopy showed the presence of some pores and channels in the blue maize starch. The electrophoretic protein profiles showed differences in the bands that correspond to the enzymes involved in the starch biosynthesis; these differences could explain the variation in morphological characteristics of blue maize starches against starch from white maize.

  9. Independent Loss of Methylthioadenosine Phosphorylase (MTAP) in Primary Cutaneous T-Cell Lymphoma.

    PubMed

    Woollard, Wesley J; Kalaivani, Nithyha P; Jones, Christine L; Roper, Catherine; Tung, Lam; Lee, Jae Jin; Thomas, Bjorn R; Tosi, Isabella; Ferreira, Silvia; Beyers, Carl Z; McKenzie, Robert C T; Butler, Rosie M; Lorenc, Anna; Whittaker, Sean J; Mitchell, Tracey J

    2016-06-01

    Methylthioadenosine phosphorylase (MTAP) and the tumor suppressor genes CDKN2A-CDKN2B are frequently deleted in malignancies. The specific role of MTAP in cutaneous T-cell lymphoma subgroups, mycosis fungoides (MF) and Sézary syndrome (SS), is unknown. In 213 skin samples from patients with MF/SS, MTAP copy number loss (34%) was more frequent than CDKN2A (12%) in all cutaneous T-cell lymphoma stages using quantitative reverse transcription PCR. Importantly, in early stage MF, MTAP loss occurred independently of CDKN2A loss in 37% of samples. In peripheral blood mononuclear cells from patients with SS, codeletion with CDKN2A occurred in 18% of samples but loss of MTAP alone was uncommon. In CD4(+) cells from SS, reduced MTAP mRNA expression correlated with MTAP copy number loss (P < 0.01) but reduced MTAP expression was also detected in the absence of copy number loss. Deep sequencing of MTAP/CDKN2A-CDKN2B loci in 77 peripheral blood mononuclear cell DNA samples from patients with SS did not show any nonsynonymous mutations, but read-depth analysis suggested focal deletions consistent with MTAP and CDKN2A copy number loss detected with quantitative reverse transcription PCR. In a cutaneous T-cell lymphoma cell line, promoter hypermethylation was shown to downregulate MTAP expression and may represent a mechanism of MTAP inactivation. In conclusion, our findings suggest that there may be selection in early stages of MF for MTAP deletion within the cutaneous tumor microenvironment.

  10. Liver as a source for thymidine phosphorylase replacement in mitochondrial neurogastrointestinal encephalomyopathy.

    PubMed

    Boschetti, Elisa; D'Alessandro, Roberto; Bianco, Francesca; Carelli, Valerio; Cenacchi, Giovanna; Pinna, Antonio D; Del Gaudio, Massimo; Rinaldi, Rita; Stanghellini, Vincenzo; Pironi, Loris; Rhoden, Kerry; Tugnoli, Vitaliano; Casali, Carlo; De Giorgio, Roberto

    2014-01-01

    Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a rare autosomal recessive mitochondrial disease associated with mutations in the nuclear TYMP gene. As a result, the thymidine phosphorylase (TP) enzyme activity is markedly reduced leading to toxic accumulation of thymidine and therefore altered mitochondrial DNA. MNGIE is characterized by severe gastrointestinal dysmotility, neurological impairment, reduced life expectancy and poor quality of life. There are limited therapeutic options for MNGIE. In the attempt to restore TP activity, allogenic hematopoietic stem cell transplantation has been used as cellular source of TP. The results of this approach on ∼ 20 MNGIE patients showed gastrointestinal and neurological improvement, although the 5-year mortality rate is about 70%. In this study we tested whether the liver may serve as an alternative source of TP. We investigated 11 patients (7M; 35-55 years) who underwent hepatic resection for focal disorders. Margins of normal liver tissue were processed to identify, quantify and localize the TP protein by Western Blot, ELISA, and immunohistochemistry, and to evaluate TYMP mRNA expression by qPCR. Western Blot identified TP in liver with a TP/GAPDH ratio of 0.9 ± 0.5. ELISA estimated TP content as 0.5 ± 0.07 ng/μg of total protein. TP was identified in both nuclei and cytoplasm of hepatocytes and sinusoidal lining cells. Finally, TYMP mRNA was expressed in the liver. Overall, our study demonstrates that the liver is an important source of TP. Orthotopic liver transplantation may be considered as a therapeutic alternative for MNGIE patients.

  11. Independent Loss of Methylthioadenosine Phosphorylase (MTAP) in Primary Cutaneous T-Cell Lymphoma.

    PubMed

    Woollard, Wesley J; Kalaivani, Nithyha P; Jones, Christine L; Roper, Catherine; Tung, Lam; Lee, Jae Jin; Thomas, Bjorn R; Tosi, Isabella; Ferreira, Silvia; Beyers, Carl Z; McKenzie, Robert C T; Butler, Rosie M; Lorenc, Anna; Whittaker, Sean J; Mitchell, Tracey J

    2016-06-01

    Methylthioadenosine phosphorylase (MTAP) and the tumor suppressor genes CDKN2A-CDKN2B are frequently deleted in malignancies. The specific role of MTAP in cutaneous T-cell lymphoma subgroups, mycosis fungoides (MF) and Sézary syndrome (SS), is unknown. In 213 skin samples from patients with MF/SS, MTAP copy number loss (34%) was more frequent than CDKN2A (12%) in all cutaneous T-cell lymphoma stages using quantitative reverse transcription PCR. Importantly, in early stage MF, MTAP loss occurred independently of CDKN2A loss in 37% of samples. In peripheral blood mononuclear cells from patients with SS, codeletion with CDKN2A occurred in 18% of samples but loss of MTAP alone was uncommon. In CD4(+) cells from SS, reduced MTAP mRNA expression correlated with MTAP copy number loss (P < 0.01) but reduced MTAP expression was also detected in the absence of copy number loss. Deep sequencing of MTAP/CDKN2A-CDKN2B loci in 77 peripheral blood mononuclear cell DNA samples from patients with SS did not show any nonsynonymous mutations, but read-depth analysis suggested focal deletions consistent with MTAP and CDKN2A copy number loss detected with quantitative reverse transcription PCR. In a cutaneous T-cell lymphoma cell line, promoter hypermethylation was shown to downregulate MTAP expression and may represent a mechanism of MTAP inactivation. In conclusion, our findings suggest that there may be selection in early stages of MF for MTAP deletion within the cutaneous tumor microenvironment. PMID:26872600

  12. Concerted action of two subunits of the functional dimer of Shewanella oneidensis MR-1 uridine phosphorylase derived from a comparison of the C212S mutant and the wild-type enzyme.

    PubMed

    Safonova, T N; Mordkovich, N N; Veiko, V P; Okorokova, N A; Manuvera, V A; Dorovatovskii, P V; Popov, V O; Polyakov, K M

    2016-02-01

    Uridine phosphorylase (UP; EC 2.4.2.3), a key enzyme in the pyrimidine-salvage pathway, catalyzes the reversible phosphorolysis of uridine to uracil and ribose 1-phosphate. The structure of the C212S mutant of uridine phosphorylase from the facultatively aerobic Gram-negative γ-proteobacterium Shewanella oneidensis MR-1 (SoUP) was determined at 1.68 Å resolution. A comparison of the structures of the mutant and the wild-type enzyme showed that one dimer in the mutant hexamer differs from all other dimers in the mutant and wild-type SoUP (both in the free form and in complex with uridine). The key difference is the `maximum open' state of one of the subunits comprising this dimer, which has not been observed previously for uridine phosphorylases. Some conformational features of the SoUP dimer that provide access of the substrate into the active site are revealed. The binding of the substrate was shown to require the concerted action of two subunits of the dimer. The changes in the three-dimensional structure induced by the C212S mutation account for the lower affinity of the mutant for inorganic phosphate, while the affinity for uridine remains unchanged.

  13. Large-scale isolation, fractionation, and purification of soluble starch-synthesizing enzymes: starch synthase and branching enzyme from potato tubers.

    PubMed

    Mukerjea, Rupendra; Falconer, Daniel J; Yoon, Seung-Heon; Robyt, John F

    2010-07-19

    Soluble starch-synthesizing enzymes, starch synthase (SSS) and starch-branching enzyme (SBE), were isolated, fractionated, and purified from white potato tubers (Solanum tuberosum) on a large scale. Five steps were used: potato tuber extract from 2 kg of peeled potatoes, two acetone precipitations, and two fractionations on a large ultrafiltration polysulfone hollow fiber 100 kDa cartridge. Three kinds of fractions were obtained: (1) mixtures of SSS and SBE; (2) SSS, free of SBE; and (3) SBE, free of SSS. Contaminating enzymes (amylase, phosphorylase, and disproportionating enzyme) and carbohydrates were absent from the 2nd acetone precipitate and from the column fractions, as judged by the Molisch test and starch triiodide test. Activity yields of 122% (300,000-400,000 units) of SSS fractions and 187% (40,000-50,000 units) of SBE fractions were routinely obtained from the cartridge. Addition of 0.04% (w/v) polyvinyl alcohol 50K and 1 mM dithiothreitol to the glycine buffer (pH 8.4) gave long-term stability and higher yields of SSS and SBE, due to activation of inactive enzymes. Several SSS and SBE fractions from the two fractionations had very high specific activities, indicating high degrees of purification. Polyacrylamide gel electrophoresis of selected SSS and SBE fractions gave two to five SSS and/or SBE activity bands, corresponding to the one to five protein bands present in the 2nd acetone precipitate.

  14. The essential role of methylthioadenosine phosphorylase in prostate cancer

    PubMed Central

    Foster, Barbara A.; Karasik, Ellen; Gillard, Bryan; Morrison, Carl; Mohler, James; Phillips, James G.; Smiraglia, Dominic J.

    2016-01-01

    Prostatic epithelial cells secrete high levels of acetylated polyamines into the prostatic lumen. This distinctive characteristic places added strain on the connected pathways, which are forced to increase metabolite production to maintain pools. The methionine salvage pathway recycles the one-carbon unit lost to polyamine biosynthesis back to the methionine cycle, allowing for replenishment of SAM pools providing a mechanism to help mitigate metabolic stress associated with high flux through these pathways. The rate-limiting enzyme involved in this process is methylthioadenosine phosphorylase (MTAP), which, although commonly deleted in many cancers, is protected in prostate cancer. We report near universal retention of MTAP expression in a panel of human prostate cancer cell lines as well as patient samples. Upon metabolic perturbation, prostate cancer cell lines upregulate MTAP and this correlates with recovery of SAM levels. Furthermore, in a mouse model of prostate cancer we find that both normal prostate and diseased prostate maintain higher SAM levels than other tissues, even under increased metabolic stress. Finally, we show that knockdown of MTAP, both genetically and pharmacologically, blocks androgen sensitive prostate cancer growth in vivo. Our findings strongly suggest that the methionine salvage pathway is a major player in homeostatic regulation of metabolite pools in prostate cancer due to their high level of flux through the polyamine biosynthetic pathway. Therefore, this pathway, and specifically the MTAP enzyme, is an attractive therapeutic target for prostate cancer. PMID:26910893

  15. Vorinostat synergises with capecitabine through upregulation of thymidine phosphorylase

    PubMed Central

    Di Gennaro, E; Piro, G; Chianese, M I; Franco, R; Cintio, A Di; Moccia, T; Luciano, A; de Ruggiero, I; Bruzzese, F; Avallone, A; Arra, C; Budillon, A

    2010-01-01

    Background: Potentiation of anticancer activity of capecitabine is required to improve its therapeutic index. In colorectal cancer (CRC) cells, we evaluated whether the histone deacetylase-inhibitor vorinostat may induce synergistic antitumour effects in combination with capecitabine by modulating the expression of thymidine phosphorylase (TP), a key enzyme in the conversion of capecitabine to 5-florouracil (5-FU), and thymidylate synthase (TS), the target of 5-FU. Methods: Expression of TP and TS was measured by real-time PCR, western blotting and immunohistochemistry. Knockdown of TP was performed by specific small interfering RNA. Antitumour activity of vorinostat was assessed in vitro in combination with the capecitabine active metabolite deoxy-5-fluorouridine (5′-DFUR) according to the Chou and Talay method and by evaluating apoptosis as well as in xenografts-bearing nude mice in combination with capecitabine. Results: Vorinostat induced both in vitro and in vivo upregulation of TP as well as downregulation of TS in cancer cells, but not in ex vivo treated peripheral blood lymphocytes. Combined treatment with vorinostat and 5′-DFUR resulted in a synergistic antiproliferative effect and increased apoptotic cell death in vitro. This latter effect was impaired in cells where TP was knocked. In vivo, vorinostat plus capecitabine potently inhibited tumour growth, increased apoptosis and prolonged survival compared with control or single-agent treatments. Conclusions: Overall, this study suggests that the combination of vorinostat and capecitabine is an innovative antitumour strategy and warrants further clinical evaluation for the treatment of CRC. PMID:21045833

  16. Thymidine phosphorylase, 2-deoxy-D-ribose and angiogenesis.

    PubMed Central

    Brown, N S; Bicknell, R

    1998-01-01

    Angiogenesis is the term used to describe the formation of new blood vessels from the existing vasculature. In order to attract new vessels, a tissue must release an endothelial-cell chemoattractant. 2-Deoxy-D-ribose is produced in vivo by the catalytic action of thymidine phosphorylase (TP) on thymidine and has recently been identified as an endothelial-cell chemoattractant and angiogenesis-inducing factor. TP, previously known only for its role in nucleotide salvage, is now known to be angiogenic. TP expression is elevated in many solid tumours and in chronically inflamed tissues, both known areas of active angiogenesis. There is evidence that TP is also involved in physiological angiogenesis such as endometrial angiogenesis during the menstrual cycle. The majority of known endothelial-cell chemoattractants are polypeptides that bind to endothelial-cell-surface receptors. In contrast, 2-deoxy-D-ribose appears to lack a cell-surface receptor. Glucose is another sugar that acts as an endothelial-cell chemoattractant. The migratory activity of glucose is blocked by ouabain. It is possible that 2-deoxy-D-ribose and glucose stimulate endothelial-cell migration via a similar mechanistic pathway. PMID:9693094

  17. Properties and functions of the storage sites of glycogen phosphorylase.

    PubMed

    Makino, Yasushi; Fujii, Yuta; Taniguchi, Motoi

    2015-06-01

    Glycogen phosphorylase (GP) is biologically active as a dimer of identical subunits. Each subunit has two distinct maltooligosaccharide binding sites: a storage site and a catalytic site. Our characterization of the properties of these sites suggested that GP activity consists of two activities: (i) binding to the glycogen molecule and (ii) phosphorolysis of the non-reducing-end glucose residues. Activity (i) is mainly due to the activities of the two storage sites, which depended on the ionic strength of the medium and were directly inhibited by cyclodextrins (CDs). Activity (i) is of benefit to GP because a high concentration of non-reducing-end glucose residues is localized on the surface of the glycogen molecule. Activity (ii), the total activity of the two catalytic sites, exhibited relatively little ionic strength dependence. Because the combined activity of (i) and (ii) is deduced using glycogen as an assay substrate, the sole activity of (ii) must be measured using small maltooligosyl-substrates. By using a very low concentration of pyridylaminated maltohexaose, we demonstrated that the GP catalytic sites are active even in the presence of CDs, and that the actions of the catalytic site and the storage site are independent of each other.

  18. Polynucleotide Phosphorylase Protects Escherichia coli against Oxidative Stress†

    PubMed Central

    Wu, Jinhua; Jiang, Zhe; Liu, Min; Gong, Xin; Wu, Shaohui; Burns, Christopher M.; Li, Zhongwei

    2009-01-01

    Escherichia coli polynucleotide phosphorylase (PNPase) primarily functions in RNA degradation. It is an exoribonuclease and integral component of the multienzyme RNA degradosome complex [Carpousis et al. (1994) Cell 76, 889]. PNPase was previously shown to specifically bind a synthetic RNA containing the oxidative lesion 8-hydroxyguanine (8-oxoG) [Hayakawa et al. (2001) Biochemistry 40, 9977], suggesting a possible role in removing oxidatively damaged RNA. Here we show that PNPase binds to RNA molecules of natural sequence that were oxidatively damaged by treatment with hydrogen peroxide (H2O2) postsynthetically. PNPase bound oxidized RNA with higher affinity than untreated RNA of the same sequence, raising the possibility that it may act against a wide variety of lesions. The importance of such a protective role is illustrated by the observation that, under conditions known to cause oxidative damage to cytoplasmic components, PNPase-deficient cells are less viable than wild-type cells. Further, when challenged with H2O2, PNPase-deficient cells accumulate 8-oxoG in cellular RNA to a greater extent than wild-type cells, suggesting that this RNase functions in minimizing oxidized RNA in vivo. Introducing the pnp gene encoding PNPase rescues defects in growth and RNA quality of the pnp mutant cells. Our results also suggest that protection against oxidative stress is an intrinsic function of PNPase because association with the RNA degradosome or with RNA helicase B (RhlB) is not required. PMID:19219992

  19. Inhibition and Structure of Toxoplasma gondii Purine Nucleoside Phosphorylase

    PubMed Central

    Donaldson, Teraya M.; Cassera, María B.; Ho, Meng-Chiao; Zhan, Chenyang; Merino, Emilio F.; Evans, Gary B.; Tyler, Peter C.; Almo, Steven C.; Schramm, Vern L.

    2014-01-01

    The intracellular pathogen Toxoplasma gondii is a purine auxotroph that relies on purine salvage for proliferation. We have optimized T. gondii purine nucleoside phosphorylase (TgPNP) stability and crystallized TgPNP with phosphate and immucillin-H, a transition-state analogue that has high affinity for the enzyme. Immucillin-H bound to TgPNP with a dissociation constant of 370 pM, the highest affinity of 11 immucillins selected to probe the catalytic site. The specificity for transition-state analogues indicated an early dissociative transition state for TgPNP. Compared to Plasmodium falciparum PNP, large substituents surrounding the 5′-hydroxyl group of inhibitors demonstrate reduced capacity for TgPNP inhibition. Catalytic discrimination against large 5′ groups is consistent with the inability of TgPNP to catalyze the phosphorolysis of 5′-methylthioinosine to hypoxanthine. In contrast to mammalian PNP, the 2′-hydroxyl group is crucial for inhibitor binding in the catalytic site of TgPNP. This first crystal structure of TgPNP describes the basis for discrimination against 5′-methylthioinosine and similarly 5′-hydroxy-substituted immucillins; structural differences reflect the unique adaptations of purine salvage pathways of Apicomplexa. PMID:24585883

  20. Neisseria meningitidis Polynucleotide Phosphorylase Affects Aggregation, Adhesion, and Virulence

    PubMed Central

    Engman, Jakob; Negrea, Aurel; Sigurlásdóttir, Sara; Geörg, Miriam; Eriksson, Jens; Eriksson, Olaspers Sara; Kuwae, Asaomi; Sjölinder, Hong

    2016-01-01

    Neisseria meningitidis autoaggregation is an important step during attachment to human cells. Aggregation is mediated by type IV pili and can be modulated by accessory pilus proteins, such as PilX, and posttranslational modifications of the major pilus subunit PilE. The mechanisms underlying the regulation of aggregation remain poorly characterized. Polynucleotide phosphorylase (PNPase) is a 3′–5′ exonuclease that is involved in RNA turnover and the regulation of small RNAs. In this study, we biochemically confirm that NMC0710 is the N. meningitidis PNPase, and we characterize its role in N. meningitidis pathogenesis. We show that deletion of the gene encoding PNPase leads to hyperaggregation and increased adhesion to epithelial cells. The aggregation induced was found to be dependent on pili and to be mediated by excessive pilus bundling. PNPase expression was induced following bacterial attachment to human cells. Deletion of PNPase led to global transcriptional changes and the differential regulation of 469 genes. We also demonstrate that PNPase is required for full virulence in an in vivo model of N. meningitidis infection. The present study shows that PNPase negatively affects aggregation, adhesion, and virulence in N. meningitidis. PMID:26930706

  1. L+-lactic acid production from starch by a novel amylolytic Lactococcus lactis subsp. lactis B84.

    PubMed

    Petrov, Kaloyan; Urshev, Zoltan; Petrova, Penka

    2008-06-01

    A new Lactococcus lactis subsp. lactis B84, capable of utilizing starch as a sole carbon source and producing L(+)-lactate, was isolated from spontaneously fermented rye sourdough. Aiming at maximum lactic acid productivity, the components of the media and the cultivation conditions were varied. In MRS-starch medium (with absence of yeast and meat extracts), at 33 degrees C, agitation 200 rpm and pH 6.0 for 6 days complete starch hydrolysis occurred and 5.5 gl(-1) lactic acid were produced from 18 gl(-1) starch. The identification of strain B84 was based on genetic criteria. Amplified ribosomal DNA restriction analysis (ARDRA), PCR with species-specific primers and sequencing of the 16S rDNA proved its species affiliation. Four genes for enzymes, involved in starch degradation were detected in B84 genome: amyL, amyY, glgP and apu, coding cytoplasmic and extracellular alpha-amylases, glycogen phosphorylase and amylopullulanase, respectively. Reverse transcription PCR experiments showed that both genes, encoding alpha-amylases (amyL and amyY) were expressed into mRNAs, whereas apu and glgP were not. Amylase activity assay was performed at different pH and temperatures. The cell-bond amylase proved to be the key enzyme, involved in the starch hydrolysis with maximum activity at 45 degrees C and pH 5.4.

  2. L+-lactic acid production from starch by a novel amylolytic Lactococcus lactis subsp. lactis B84.

    PubMed

    Petrov, Kaloyan; Urshev, Zoltan; Petrova, Penka

    2008-06-01

    A new Lactococcus lactis subsp. lactis B84, capable of utilizing starch as a sole carbon source and producing L(+)-lactate, was isolated from spontaneously fermented rye sourdough. Aiming at maximum lactic acid productivity, the components of the media and the cultivation conditions were varied. In MRS-starch medium (with absence of yeast and meat extracts), at 33 degrees C, agitation 200 rpm and pH 6.0 for 6 days complete starch hydrolysis occurred and 5.5 gl(-1) lactic acid were produced from 18 gl(-1) starch. The identification of strain B84 was based on genetic criteria. Amplified ribosomal DNA restriction analysis (ARDRA), PCR with species-specific primers and sequencing of the 16S rDNA proved its species affiliation. Four genes for enzymes, involved in starch degradation were detected in B84 genome: amyL, amyY, glgP and apu, coding cytoplasmic and extracellular alpha-amylases, glycogen phosphorylase and amylopullulanase, respectively. Reverse transcription PCR experiments showed that both genes, encoding alpha-amylases (amyL and amyY) were expressed into mRNAs, whereas apu and glgP were not. Amylase activity assay was performed at different pH and temperatures. The cell-bond amylase proved to be the key enzyme, involved in the starch hydrolysis with maximum activity at 45 degrees C and pH 5.4. PMID:18456109

  3. Structure of purine nucleoside phosphorylase (DeoD) from Bacillus anthracis

    SciTech Connect

    Grenha, Rosa; Levdikov, Vladimir M.; Fogg, Mark J.; Blagova, Elena V.; Brannigan, James A. Wilkinson, Anthony J.; Wilson, Keith S.

    2005-05-01

    The crystal structure of purine nucleoside phosphorylase (DeoD) from B. anthracis was solved by X-ray crystallography using molecular replacement and refined at a resolution of 2.24 Å. Protein structures from the causative agent of anthrax (Bacillus anthracis) are being determined as part of a structural genomics programme. Amongst initial candidates for crystallographic analysis are enzymes involved in nucleotide biosynthesis, since these are recognized as potential targets in antibacterial therapy. Purine nucleoside phosphorylase is a key enzyme in the purine-salvage pathway. The crystal structure of purine nucleoside phosphorylase (DeoD) from B. anthracis has been solved by molecular replacement at 2.24 Å resolution and refined to an R factor of 18.4%. This is the first report of a DeoD structure from a Gram-positive bacterium.

  4. Glycogen phosphorylase sequences from the amitochondriate protists, Trichomonas vaginalis, Mastigamoeba balamuthi, Entamoeba histolytica and Giardia intestinalis.

    PubMed

    Wu, Gang; Müller, Miklós

    2003-01-01

    Glycogen phosphorylase genes or messages from four amitochondriate eukaryotes, Trichomonas vaginalis, Mastigamoeba balamuthi, Entamoeba histolytica (two genes) and Giardia intestinalis, have been isolated and sequenced. The sequences of the amitochondriate protist enzymes appear to share a most recent common ancestor. The clade containing these sequences is closest to that of another protist, the slime mold (Dictyostelium discoideum), and is more closely related to fungal and plant phosphorylases than to mammalian and eubacterial homologs. Structure-based amino acid alignment shows conservation of the residues and domains involved in catalysis and allosteric regulation by glucose 6-phosphate but high divergence at domains involved in phosphorylation-dependent regulation and AMP binding in fungi and animals. Protist phosphorylases, as their prokaryotic and plant counterparts, are probably not regulated by phosphorylation.

  5. Characterization of starch nanoparticles

    NASA Astrophysics Data System (ADS)

    Szymońska, J.; Targosz-Korecka, M.; Krok, F.

    2009-01-01

    Nanomaterials already attract great interest because of their potential applications in technology, food science and medicine. Biomaterials are biodegradable and quite abundant in nature, so they are favoured over synthetic polymer based materials. Starch as a nontoxic, cheap and renewable raw material is particularly suitable for preparation of nanoparticles. In the paper, the structure and some physicochemical properties of potato and cassava starch particles of the size between 50 to 100 nm, obtained by mechanical treatment of native starch, were presented. We demonstrated, with the aim of the Scanning Electron Microscopy (SEM) and the non-contact Atomic Force Microscopy (nc-AFM), that the shape and dimensions of the obtained nanoparticles both potato and cassava starch fit the blocklets - previously proposed as basic structural features of native starch granules. This observation was supported by aqueous solubility and swelling power of the particles as well as their iodine binding capacity similar to those for amylopectin-type short branched polysaccharide species. Obtained results indicated that glycosidic bonds of the branch linkage points in the granule amorphous lamellae might be broken during the applied mechanical treatment. Thus the released amylopectin clusters could escape out of the granules. The starch nanoparticles, for their properties qualitatively different from those of native starch granules, could be utilized in new applications.

  6. Starch Metabolism in Arabidopsis

    PubMed Central

    Streb, Sebastian; Zeeman, Samuel C.

    2012-01-01

    Starch is the major non-structural carbohydrate in plants. It serves as an important store of carbon that fuels plant metabolism and growth when they are unable to photosynthesise. This storage can be in leaves and other green tissues, where it is degraded during the night, or in heterotrophic tissues such as roots, seeds and tubers, where it is stored over longer time periods. Arabidopsis accumulates starch in many of its tissues, but mostly in its leaves during the day. It has proven to be a powerful genetic system for discovering how starch is synthesised and degraded, and new proteins and processes have been discovered. Such work has major significance for our starch crops, whose yield and quality could be improved by the application of this knowledge. Research into Arabidopsis starch metabolism has begun to reveal how its daily turnover is integrated into the rest of metabolism and adapted to the environmental conditions. Furthermore, Arabidopsis mutant lines deficient in starch metabolism have been employed as tools to study other biological processes ranging from sugar sensing to gravitropism and flowering time control. This review gives a detailed account of the use of Arabidopsis to study starch metabolism. It describes the major discoveries made and presents an overview of our understanding today, together with some as-yet unresolved questions. PMID:23393426

  7. Characterization and crystal structure determination of β-1,2-mannobiose phosphorylase from Listeria innocua.

    PubMed

    Tsuda, Tomohiro; Nihira, Takanori; Chiku, Kazuhiro; Suzuki, Erika; Arakawa, Takatoshi; Nishimoto, Mamoru; Kitaoka, Motomitsu; Nakai, Hiroyuki; Fushinobu, Shinya

    2015-12-21

    Glycoside hydrolase family 130 consists of phosphorylases and hydrolases for β-mannosides. Here, we characterized β-1,2-mannobiose phosphorylase from Listeria innocua (Lin0857) and determined its crystal structures complexed with β-1,2-linked mannooligosaccharides. β-1,2-Mannotriose was bound in a U-shape, interacting with a phosphate analog at both ends. Lin0857 has a unique dimer structure connected by a loop, and a significant open-close loop displacement was observed for substrate entry. A long loop, which is exclusively present in Lin0857, covers the active site to limit the pocket size. A structural basis for substrate recognition and phosphorolysis was provided.

  8. An evaluation of indirubin analogues as phosphorylase kinase inhibitors.

    PubMed

    Begum, Jaida; Skamnaki, Vassiliki T; Moffatt, Colin; Bischler, Nicolas; Sarrou, Josephine; Skaltsounis, Alexios-Leandros; Leonidas, Demetres D; Oikonomakos, Nikos G; Hayes, Joseph M

    2015-09-01

    Phosphorylase kinase (PhK) has been linked with a number of conditions such as glycogen storage diseases, psoriasis, type 2 diabetes and more recently, cancer (Camus et al., 2012 [6]). However, with few reported structural studies on PhK inhibitors, this hinders a structure based drug design approach. In this study, the inhibitory potential of 38 indirubin analogues have been investigated. 11 of these ligands had IC50 values in the range 0.170-0.360μM, with indirubin-3'-acetoxime (1c) the most potent. 7-Bromoindirubin-3'-oxime (13b), an antitumor compound which induces caspase-independent cell-death (Ribas et al., 2006 [20]) is revealed as a specific inhibitor of PhK (IC50=1.8μM). Binding assay experiments performed using both PhK-holo and PhK-γtrnc confirmed the inhibitory effects to arise from binding at the kinase domain (γ subunit). High level computations using QM/MM-PBSA binding free energy calculations were in good agreement with experimental binding data, as determined using statistical analysis, and support binding at the ATP-binding site. The value of a QM description for the binding of halogenated ligands exhibiting σ-hole effects is highlighted. A new statistical metric, the 'sum of the modified logarithm of ranks' (SMLR), has been defined which measures performance of a model for both the "early recognition" (ranking earlier/higher) of active compounds and their relative ordering by potency. Through a detailed structure activity relationship analysis considering other kinases (CDK2, CDK5 and GSK-3α/β), 6'(Z) and 7(L) indirubin substitutions have been identified to achieve selective PhK inhibition. The key PhK binding site residues involved can also be targeted using other ligand scaffolds in future work. PMID:26364215

  9. An evaluation of indirubin analogues as phosphorylase kinase inhibitors.

    PubMed

    Begum, Jaida; Skamnaki, Vassiliki T; Moffatt, Colin; Bischler, Nicolas; Sarrou, Josephine; Skaltsounis, Alexios-Leandros; Leonidas, Demetres D; Oikonomakos, Nikos G; Hayes, Joseph M

    2015-09-01

    Phosphorylase kinase (PhK) has been linked with a number of conditions such as glycogen storage diseases, psoriasis, type 2 diabetes and more recently, cancer (Camus et al., 2012 [6]). However, with few reported structural studies on PhK inhibitors, this hinders a structure based drug design approach. In this study, the inhibitory potential of 38 indirubin analogues have been investigated. 11 of these ligands had IC50 values in the range 0.170-0.360μM, with indirubin-3'-acetoxime (1c) the most potent. 7-Bromoindirubin-3'-oxime (13b), an antitumor compound which induces caspase-independent cell-death (Ribas et al., 2006 [20]) is revealed as a specific inhibitor of PhK (IC50=1.8μM). Binding assay experiments performed using both PhK-holo and PhK-γtrnc confirmed the inhibitory effects to arise from binding at the kinase domain (γ subunit). High level computations using QM/MM-PBSA binding free energy calculations were in good agreement with experimental binding data, as determined using statistical analysis, and support binding at the ATP-binding site. The value of a QM description for the binding of halogenated ligands exhibiting σ-hole effects is highlighted. A new statistical metric, the 'sum of the modified logarithm of ranks' (SMLR), has been defined which measures performance of a model for both the "early recognition" (ranking earlier/higher) of active compounds and their relative ordering by potency. Through a detailed structure activity relationship analysis considering other kinases (CDK2, CDK5 and GSK-3α/β), 6'(Z) and 7(L) indirubin substitutions have been identified to achieve selective PhK inhibition. The key PhK binding site residues involved can also be targeted using other ligand scaffolds in future work.

  10. The Starch Granule-Associated Protein EARLY STARVATION1 Is Required for the Control of Starch Degradation in Arabidopsis thaliana Leaves[OPEN

    PubMed Central

    Feike, Doreen; Seung, David; Graf, Alexander; Bischof, Sylvain; Ellick, Tamaryn; Coiro, Mario; Soyk, Sebastian; Eicke, Simona; Mettler-Altmann, Tabea; Lu, Kuan Jen; Trick, Martin; Zeeman, Samuel C.

    2016-01-01

    To uncover components of the mechanism that adjusts the rate of leaf starch degradation to the length of the night, we devised a screen for mutant Arabidopsis thaliana plants in which starch reserves are prematurely exhausted. The mutation in one such mutant, named early starvation1 (esv1), eliminates a previously uncharacterized protein. Starch in mutant leaves is degraded rapidly and in a nonlinear fashion, so that reserves are exhausted 2 h prior to dawn. The ESV1 protein and a similar uncharacterized Arabidopsis protein (named Like ESV1 [LESV]) are located in the chloroplast stroma and are also bound into starch granules. The region of highest similarity between the two proteins contains a series of near-repeated motifs rich in tryptophan. Both proteins are conserved throughout starch-synthesizing organisms, from angiosperms and monocots to green algae. Analysis of transgenic plants lacking or overexpressing ESV1 or LESV, and of double mutants lacking ESV1 and another protein necessary for starch degradation, leads us to propose that these proteins function in the organization of the starch granule matrix. We argue that their misexpression affects starch degradation indirectly, by altering matrix organization and, thus, accessibility of starch polymers to starch-degrading enzymes. PMID:27207856

  11. The Starch Granule-Associated Protein EARLY STARVATION1 Is Required for the Control of Starch Degradation in Arabidopsis thaliana Leaves.

    PubMed

    Feike, Doreen; Seung, David; Graf, Alexander; Bischof, Sylvain; Ellick, Tamaryn; Coiro, Mario; Soyk, Sebastian; Eicke, Simona; Mettler-Altmann, Tabea; Lu, Kuan Jen; Trick, Martin; Zeeman, Samuel C; Smith, Alison M

    2016-06-01

    To uncover components of the mechanism that adjusts the rate of leaf starch degradation to the length of the night, we devised a screen for mutant Arabidopsis thaliana plants in which starch reserves are prematurely exhausted. The mutation in one such mutant, named early starvation1 (esv1), eliminates a previously uncharacterized protein. Starch in mutant leaves is degraded rapidly and in a nonlinear fashion, so that reserves are exhausted 2 h prior to dawn. The ESV1 protein and a similar uncharacterized Arabidopsis protein (named Like ESV1 [LESV]) are located in the chloroplast stroma and are also bound into starch granules. The region of highest similarity between the two proteins contains a series of near-repeated motifs rich in tryptophan. Both proteins are conserved throughout starch-synthesizing organisms, from angiosperms and monocots to green algae. Analysis of transgenic plants lacking or overexpressing ESV1 or LESV, and of double mutants lacking ESV1 and another protein necessary for starch degradation, leads us to propose that these proteins function in the organization of the starch granule matrix. We argue that their misexpression affects starch degradation indirectly, by altering matrix organization and, thus, accessibility of starch polymers to starch-degrading enzymes. PMID:27207856

  12. Resistant starches and health.

    PubMed

    Kendall, Cyril W C; Emam, Azadeh; Augustin, Livia S A; Jenkins, David J A

    2004-01-01

    It was initially hypothesized that resistant starches, i.e., starch that enters the colon, would have protective effects on chronic colonic diseases, including reduction of colon cancer risk and in the treatment of ulcerative colitis. Recent studies have confirmed the ability of resistant starch to increase fecal bulk, increase the molar ratio of butyrate in relation to other short-chain fatty acids, and dilute fecal bile acids. However the ability of resistant starch to reduce luminal concentrations of compounds that are damaging to the colonic mucosa, including fecal ammonia, phenols, and N-nitroso compounds, still requires clear demonstration. As such, the effectiveness of resistant starch in preventing or treating colonic diseases remains to be assessed. Nevertheless, there is a fraction of what has been termed resistant (RS1) starch, which enters the colon and acts as slowly digested or lente carbohydrate in the small intestine. Foods in this class are low glycemic index and have been shown to reduce the risk of chronic disease. They have been associated with systemic physiological effects such as reduced postprandial insulin levels and higher HDL cholesterol levels. Consumption of low glycemic index foods has been shown to be related to reductions in risk of coronary heart disease and Type 2 diabetes. Type 2 diabetes has in turn been related to a higher risk of colon cancer. If carbohydrates have a protective role in colon cancer prevention this may lie partly in the systemic effects of low glycemic index foods. The colonic advantages of different carbohydrates, varying in their glycemic index and resistant starch content, therefore, remain to be determined. However, as recent positive research findings continue to mount, there is reason for optimism over the possible health advantages of those resistant starches, which are slowly digested in the small intestine. PMID:15287678

  13. Methylthioinosine Phosphorylase from Pseudomonas aeruginosa. Structure and Annotation of a Novel Enzyme in Quorum Sensing†

    PubMed Central

    Guan, Rong; Ho, Meng-Chiao; Almo, Steven C.; Schramm, Vern L.

    2011-01-01

    The PA3004 gene of Pseudomonas aeruginosa PAO1 was originally annotated as a 5’-methylthioadenosine phosphorylase (MTAP). However, the PA3004 encoded protein uses 5’-methylthioinosine (MTI) as a preferred substrate and represents the only known example of a specific MTI phosphorylase (MTIP). MTIP does not utilize 5’-methylthioadenosine (MTA). Inosine is a weak substrate with a kcat/Km value 290-fold less than MTI and is the second best substrate identified. The crystal structure of P. aeruginosa MTIP (PaMTIP) in complex with hypoxanthine was determined to 2.8 Å resolution and revealed a three-fold symmetric homotrimer. The methylthioribose and phosphate binding regions of PaMTIP are similar to MTAPs, and the purine binding region is similar to that of purine nucleoside phosphorylases (PNPs). The catabolism of MTA in P. aeruginosa involves deamination to MTI and phosphorolysis to hypoxanthine (MTA → MTI → hypoxanthine). This pathway also exists in Plasmodium falciparum, where the purine nucleoside phosphorylase (PfPNP) acts on both inosine and MTI. Three tight-binding transition state analogue inhibitors of PaMTIP are identified with dissociation constants in the picomolar range. Inhibitor specificity suggests an early dissociative transition state for PaMTIP. Quorum sensing molecules are associated with MTA metabolism in bacterial pathogens suggesting PaMTIP as a potential therapeutic target. PMID:21197954

  14. Regulation of glycogen synthase and phosphorylase during recovery from high-intensity exercise in the rat.

    PubMed Central

    Bräu, L; Ferreira, L D; Nikolovski, S; Raja, G; Palmer, T N; Fournier, P A

    1997-01-01

    The aim of this study was to determine the role of the phosphorylation state of glycogen synthase and glycogen phosphorylase in the regulation of muscle glycogen repletion in fasted animals recovering from high-intensity exercise. Groups of rats were swum to exhaustion and allowed to recover for up to 120 min without access to food. Swimming to exhaustion caused substantial glycogen breakdown and lactate accumulation in the red, white and mixed gastrocnemius muscles, whereas the glycogen content in the soleus muscle remained stable. During the first 40 min of recovery, significant repletion of glycogen occurred in all muscles examined except the soleus muscle. At the onset of recovery, the activity ratios and fractional velocities of glycogen synthase in the red, white and mixed gastrocnemius muscles were higher than basal, but returned to pre-exercise levels within 20 min after exercise. In contrast, after exercise the activity ratios of glycogen phosphorylase in the same muscles were lower than basal, and increased to pre-exercise levels within 20 min. This pattern of changes in glycogen synthase and phosphorylase activities, never reported before, suggests that the integrated regulation of the phosphorylation state of both glycogen synthase and phosphorylase might be involved in the control of glycogen deposition after high-intensity exercise. PMID:9078277

  15. Polymer phosphorylases: clues to the emergence of non-replicative and replicative polymers.

    PubMed

    Freire, Miguel Angel

    2011-12-01

    Polymer formation is arguably one of the essential factors that allowed the emergence, stabilisation and spread of life on Earth. Consequently, studies concerning biopolymers could shed light on the origins of life itself. Of particular interest are RNA and polysaccharide polymers, the archetypes of the contrasting proposed evolutionary scenarios and their respective polymerases. Nucleic acid polymerases were hypothesised, before their discovery, to have a functional similarity with glycogen phosphorylase. Further identification and characterisation of nucleic acid polymerases; particularly of polynucleotide phosphorylase (PNPase), provided experimental evidence for the initial premise. Once discovered, frequent similarities were found between PNPase and glycogen phosphorylase, in terms of catalytic features and biochemical properties. As a result, PNPase was seen as a model of primitive polymerase and used in laboratory precellular systems. Paradoxically, however, these similarities were not sufficient as an argument in favour of an ancestral common polymerisation mechanism prior to polysaccharides and polyribonucleotides. Here we present an overview of the common features shared by polymer phosphorylases, with new proposals for the emergence of polysaccharide and RNA polymers.

  16. In vitro digestibility of banana starch cookies.

    PubMed

    Bello-Pérez, Luis A; Sáyago-Ayerdi, Sonia G; Méndez-Montealvo, Guadalupe; Tovar, Juscelino

    2004-01-01

    Banana starch was isolated and used for preparation of two types of cookies. Chemical composition and digestibility tests were carried out on banana starch and the food products, and these results were compared with corn starch. Ash, protein, and fat levels in banana starch were higher than in corn starch. The high ash amount in banana starch could be due to the potassium content present in this fruit. Proximal analysis was similar between products prepared with banana starch and those based on corn starch. The available starch content of the banana starch preparation was 60% (dmb). The cookies had lower available starch than the starches while banana starch had lower susceptibility to the in vitro alpha-amylolysis reaction. Banana starch and its products had higher resistant starch levels than those made with corn starch.

  17. Anopheles gambiae Purine Nucleoside Phosphorylase: Catalysis, Structure, and Inhibition

    SciTech Connect

    Taylor,E.; Rinaldo-Matthis, A.; Li, L.; Ghanem, M.; Hazleton, K.; Cassera, M.; Almo, S.; Schramm, V.

    2007-01-01

    The purine salvage pathway of Anopheles gambiae, a mosquito that transmits malaria, has been identified in genome searches on the basis of sequence homology with characterized enzymes. Purine nucleoside phosphorylase (PNP) is a target for the development of therapeutic agents in humans and purine auxotrophs, including malarial parasites. The PNP from Anopheles gambiae (AgPNP) was expressed in Escherichia coli and compared to the PNPs from Homo sapiens (HsPNP) and Plasmodium falciparum (PfPNP). AgPNP has kcat values of 54 and 41 s-1 for 2'-deoxyinosine and inosine, its preferred substrates, and 1.0 s-1 for guanosine. However, the chemical step is fast for AgPNP at 226 s-1 for guanosine in pre-steady-state studies. 5'-Deaza-1'-aza-2'-deoxy-1'-(9-methylene)-Immucillin-H (DADMe-ImmH) is a transition-state mimic for a 2'-deoxyinosine ribocation with a fully dissociated N-ribosidic bond and is a slow-onset, tight-binding inhibitor with a dissociation constant of 3.5 pM. This is the tightest-binding inhibitor known for any PNP, with a remarkable Km/Ki* of 5.4 x 107, and is consistent with enzymatic transition state predictions of enhanced transition-state analogue binding in enzymes with enhanced catalytic efficiency. Deoxyguanosine is a weaker substrate than deoxyinosine, and DADMe-Immucillin-G is less tightly bound than DADMe-ImmH, with a dissociation constant of 23 pM for AgPNP as compared to 7 pM for HsPNP. The crystal structure of AgPNP was determined in complex with DADMe-ImmH and phosphate to a resolution of 2.2 Angstroms to reveal the differences in substrate and inhibitor specificity. The distance from the N1' cation to the phosphate O4 anion is shorter in the AgPNP{center_dot}DADMe-ImmH{center_dot}PO4 complex than in HsPNP{center_dot}DADMe-ImmH{center_dot}SO4, offering one explanation for the stronger inhibitory effect of DADMe-ImmH for AgPNP.

  18. Starch mutants of Chlamydomonas

    SciTech Connect

    Berry-Lowe, S.L.; Schmidt, G.W. )

    1990-05-01

    Wild type Chlamydomonas accumulates starch and triglycerides when grown under nitrogen limiting conditions. Toward elucidation of the mechanisms for control of starch biosynthesis, we isolated mutants impaired int he accumulation of storage carbohydrates. Chlamydomonas reinhardtii (strain ya-12) was mutagenized by UV irradiation and colonies were screened by iodine staining after growth in darkness. Mutants, denoted ais for altered in iodine staining, have been characterized by electron microscopy and assays for starch synthease, ADPG-pyrophosphorylase, phosphoglucose isomerase (PGI), phosphoglucomutase and fructose 1,6-bisphosphatase, and amylase activities. Transcript analysis of wild type and mutant RNAs with PGI, ADPG-pyrophosphorylase, and waxy probes have also been carried out. No deficiencies of any of these components have been detected. Furthermore, long-term cultures of ya-12 and ais-1d in nitrogen-limited chemostats have been studied; starch also does not accumulate in ais-1d under these conditions. Thus, the lesion affects an essential factor of unknown identity that is required for starch synthesis.

  19. Comparison of pasting and gel stabilities of waxy and normal starches from potato, maize, and rice with those of a novel waxy cassava starch under thermal, chemical, and mechanical stress.

    PubMed

    Sánchez, Teresa; Dufour, Dominique; Moreno, Isabel Ximena; Ceballos, Hernán

    2010-04-28

    Functional properties of normal and waxy starches from maize, rice, potato, and cassava as well as the modified waxy maize starch COLFLO 67 were compared. The main objective of this study is to position the recently discovered spontaneous mutation for amylose-free cassava starch in relation to the other starches with well-known characteristics. Paste clarity, wavelength of maximum absorption (lambda(max)), pasting properties, swelling power, solubility, and dispersed volume fraction measurements and gel stability (acid and alkaline resistance, shear, refrigeration, and freeze/thaw stability) were evaluated in the different types and sources of starch included in this study. lambda(max) in the waxy cassava starch was reduced considerably in comparison with that of normal cassava starch (535 vs 592 nm). RVA peak viscosity of waxy cassava starch was larger than in normal cassava starch (1119 vs 937 cP) and assumed a position intermediate between the waxy potato and maize starches. Acid, alkaline, and shear stability of waxy cassava starch were similar to normal cassava except for alkaline pH, at which it showed a low effect. Gels from normal root and tuber starches after refrigeration and freeze/thaw had lower syneresis than cereal starches. Gels from waxy starches (except for potato) did not present any syneresis after 5 weeks of storage at 4 degrees C. Waxy cassava starch was the only one not showing any syneresis after 5 weeks of storage at -20 degrees C. Natural waxy cassava starch is, therefore, a promising ingredient to formulate refrigerated or frozen food. PMID:20356303

  20. Comparison of pasting and gel stabilities of waxy and normal starches from potato, maize, and rice with those of a novel waxy cassava starch under thermal, chemical, and mechanical stress.

    PubMed

    Sánchez, Teresa; Dufour, Dominique; Moreno, Isabel Ximena; Ceballos, Hernán

    2010-04-28

    Functional properties of normal and waxy starches from maize, rice, potato, and cassava as well as the modified waxy maize starch COLFLO 67 were compared. The main objective of this study is to position the recently discovered spontaneous mutation for amylose-free cassava starch in relation to the other starches with well-known characteristics. Paste clarity, wavelength of maximum absorption (lambda(max)), pasting properties, swelling power, solubility, and dispersed volume fraction measurements and gel stability (acid and alkaline resistance, shear, refrigeration, and freeze/thaw stability) were evaluated in the different types and sources of starch included in this study. lambda(max) in the waxy cassava starch was reduced considerably in comparison with that of normal cassava starch (535 vs 592 nm). RVA peak viscosity of waxy cassava starch was larger than in normal cassava starch (1119 vs 937 cP) and assumed a position intermediate between the waxy potato and maize starches. Acid, alkaline, and shear stability of waxy cassava starch were similar to normal cassava except for alkaline pH, at which it showed a low effect. Gels from normal root and tuber starches after refrigeration and freeze/thaw had lower syneresis than cereal starches. Gels from waxy starches (except for potato) did not present any syneresis after 5 weeks of storage at 4 degrees C. Waxy cassava starch was the only one not showing any syneresis after 5 weeks of storage at -20 degrees C. Natural waxy cassava starch is, therefore, a promising ingredient to formulate refrigerated or frozen food.

  1. Biological Implications in Cassava for the Production of Amylose-Free Starch: Impact on Root Yield and Related Traits

    PubMed Central

    Karlström, Amanda; Calle, Fernando; Salazar, Sandra; Morante, Nelson; Dufour, Dominique; Ceballos, Hernán

    2016-01-01

    Cassava (Manihot esculenta, Crantz) is an important food security crop, but it is becoming an important raw material for different industrial applications. Cassava is the second most important source of starch worldwide. Novel starch properties are of interest to the starch industry, and one them is the recently identified amylose-free (waxy) cassava starch. Waxy mutants have been found in different crops and have been often associated with a yield penalty. There are ongoing efforts to develop commercial cassava varieties with amylose-free starch. However, little information is available regarding the biological and agronomic implications of starch mutations in cassava, nor in other root and tuber crops. In this study, siblings from eight full-sib families, segregating for the waxy trait, were used to determine if the mutation has implications for yield, dry matter content (DMC) and harvest index in cassava. A total of 87 waxy and 87 wild-type starch genotypes from the eight families were used in the study. The only significant effect of starch type was on DMC (p < 0.01), with waxy clones having a 0.8% lower content than their wild type counterparts. There was no effect of starch type on fresh root yield (FRY), adjusted FRY and harvest index. It is not clear if lower DMC is a pleiotropic effect of the waxy starch mutation or else the result of linked genes introgressed along with the mutation. It is expected that commercial waxy cassava varieties will have competitive FRYs but special efforts will be required to attain adequate DMCs. This study contributes to the limited knowledge available of the impact of starch mutations on the agronomic performance of root and tuber crops. PMID:27242813

  2. Biological Implications in Cassava for the Production of Amylose-Free Starch: Impact on Root Yield and Related Traits.

    PubMed

    Karlström, Amanda; Calle, Fernando; Salazar, Sandra; Morante, Nelson; Dufour, Dominique; Ceballos, Hernán

    2016-01-01

    Cassava (Manihot esculenta, Crantz) is an important food security crop, but it is becoming an important raw material for different industrial applications. Cassava is the second most important source of starch worldwide. Novel starch properties are of interest to the starch industry, and one them is the recently identified amylose-free (waxy) cassava starch. Waxy mutants have been found in different crops and have been often associated with a yield penalty. There are ongoing efforts to develop commercial cassava varieties with amylose-free starch. However, little information is available regarding the biological and agronomic implications of starch mutations in cassava, nor in other root and tuber crops. In this study, siblings from eight full-sib families, segregating for the waxy trait, were used to determine if the mutation has implications for yield, dry matter content (DMC) and harvest index in cassava. A total of 87 waxy and 87 wild-type starch genotypes from the eight families were used in the study. The only significant effect of starch type was on DMC (p < 0.01), with waxy clones having a 0.8% lower content than their wild type counterparts. There was no effect of starch type on fresh root yield (FRY), adjusted FRY and harvest index. It is not clear if lower DMC is a pleiotropic effect of the waxy starch mutation or else the result of linked genes introgressed along with the mutation. It is expected that commercial waxy cassava varieties will have competitive FRYs but special efforts will be required to attain adequate DMCs. This study contributes to the limited knowledge available of the impact of starch mutations on the agronomic performance of root and tuber crops. PMID:27242813

  3. Biological Implications in Cassava for the Production of Amylose-Free Starch: Impact on Root Yield and Related Traits.

    PubMed

    Karlström, Amanda; Calle, Fernando; Salazar, Sandra; Morante, Nelson; Dufour, Dominique; Ceballos, Hernán

    2016-01-01

    Cassava (Manihot esculenta, Crantz) is an important food security crop, but it is becoming an important raw material for different industrial applications. Cassava is the second most important source of starch worldwide. Novel starch properties are of interest to the starch industry, and one them is the recently identified amylose-free (waxy) cassava starch. Waxy mutants have been found in different crops and have been often associated with a yield penalty. There are ongoing efforts to develop commercial cassava varieties with amylose-free starch. However, little information is available regarding the biological and agronomic implications of starch mutations in cassava, nor in other root and tuber crops. In this study, siblings from eight full-sib families, segregating for the waxy trait, were used to determine if the mutation has implications for yield, dry matter content (DMC) and harvest index in cassava. A total of 87 waxy and 87 wild-type starch genotypes from the eight families were used in the study. The only significant effect of starch type was on DMC (p < 0.01), with waxy clones having a 0.8% lower content than their wild type counterparts. There was no effect of starch type on fresh root yield (FRY), adjusted FRY and harvest index. It is not clear if lower DMC is a pleiotropic effect of the waxy starch mutation or else the result of linked genes introgressed along with the mutation. It is expected that commercial waxy cassava varieties will have competitive FRYs but special efforts will be required to attain adequate DMCs. This study contributes to the limited knowledge available of the impact of starch mutations on the agronomic performance of root and tuber crops.

  4. 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.

  5. Starch biosynthesis: experiments on how starch granules grow in vivo.

    PubMed

    Mukerjea, Romila; Mukerjea, Rupendra; Robyt, John F

    2009-01-01

    Four varieties of starch granules from potato, wheat, maize, and rice were fractionated into homogeneous 10-microm-sized ranges. The size with the largest amount of granules was reacted with ADP-[(14)C]Glc, washed, and peeled into 7-9 layers, using a controlled peeling process, involving 90:10 volume proportions of Me(2)SO-H(2)O at 10 degrees C. All of the starches showed biosynthesis of starch throughout the granules. Starch synthase activities were determined for each of the layers. Three of the starches had a relatively large amount of synthase activity in the second layer, with only a small amount in the first layer. Potato starch had the largest amount of activity in the first layer. Starch synthase activity was found to alternate between higher and lower activities throughout all of the varieties of granules, showing that the synthesis was not uniform and also was not exclusively occurring at the surface of the starch granules, which had previously been hypothesized. From these results and our previous studies on the mechanism of starch chain elongation by the addition of d-glucose to the reducing end of a growing chain that is covalently attached to the active site of starch synthase, a hypothesis is proposed for how starch granules grow in vivo.

  6. Starch phosphorylation: a new front line in starch research.

    PubMed

    Blennow, Andreas; Nielsen, Tom H; Baunsgaard, Lone; Mikkelsen, René; Engelsen, Søren B

    2002-10-01

    Starch is the primary energy reserve in higher plants and is, after cellulose, the second most abundant carbohydrate in the biosphere. It is also the most important energy source in the human diet and, being a biodegradable polymer with well-defined chemical properties, has an enormous potential as a versatile renewable resource. The only naturally occurring covalent modification of starch is phosphorylation. Starch phosphate esters were discovered a century ago but were long regarded as a curiosity, receiving little attention. Indeed, the mechanism for starch phosphorylation remained completely unknown until recently. The starch-phosphorylating enzyme is an alpha-glucan water dikinase. It is now known that starch phosphorylation plays a central role in starch metabolism.

  7. The Characterization of Modified Starch Branching Enzymes: Toward the Control of Starch Chain-Length Distributions

    PubMed Central

    Li, Cheng; Wu, Alex Chi; Go, Rob Marc; Malouf, Jacob; Turner, Mark S.; Malde, Alpeshkumar K.; Mark, Alan E.; Gilbert, Robert G.

    2015-01-01

    Starch is a complex branched glucose polymer whose branch molecular weight distribution (the chain-length distribution, CLD) influences nutritionally important properties such as digestion rate. Chain-stopping in starch biosynthesis is by starch branching enzyme (SBE). Site-directed mutagenesis was used to modify SBEIIa from Zea mays (mSBEIIa) to produce mutants, each differing in a single conserved amino-acid residue. Products at different times from in vitro branching were debranched and the time evolution of the CLD measured by size-exclusion chromatography. The results confirm that Tyr352, Glu513, and Ser349 are important for mSBEIIa activity while Arg456 is important for determining the position at which the linear glucan is cut. The mutant mSBEIIa enzymes have different activities and suggest the length of the transferred chain can be varied by mutation. The work shows analysis of the molecular weight distribution can yield information regarding the enzyme branching sites useful for development of plants yielding starch with improved functionality. PMID:25874689

  8. Purification, crystallization, and preliminary X-ray diffraction study of purine nucleoside phosphorylase from E. coli

    SciTech Connect

    Abramchik, Yu. A. Timofeev, V. I. Zhukhlistova, N. E.; Muravieva, T. I.; Esipov, R. S.; Kuranova, I. P.

    2015-07-15

    Crystals of E. coli purine nucleoside phosphorylase were grown in microgravity by the capillary counter-diffusion method through a gel layer. The X-ray diffraction data set suitable for the determination of the three-dimensional structure at atomic resolution was collected from one crystal at the Spring-8 synchrotron facility to 0.99 Å resolution. The crystals belong to sp. gr. P2{sub 1} and have the following unit-cell parameters: a = 74.1 Å, b = 110.2 Å, c = 88.2 Å, α = γ = 90°, β = 111.08°. The crystal contains six subunits of the enzyme comprising a hexamer per asymmetric unit. The hexamer is the biological active form of E. coli. purine nucleoside phosphorylase.

  9. Three-dimensional structure of E. Coli purine nucleoside phosphorylase at 0.99 Å resolution

    NASA Astrophysics Data System (ADS)

    Timofeev, V. I.; Abramchik, Yu. A.; Zhukhlistova, N. E.; Muravieva, T. I.; Esipov, R. S.; Kuranova, I. P.

    2016-03-01

    Purine nucleoside phosphorylases (PNPs) catalyze the reversible phosphorolysis of nucleosides and are key enzymes involved in nucleotide metabolism. They are essential for normal cell function and can catalyze the transglycosylation. Crystals of E. coli PNP were grown in microgravity by the capillary counterdiffusion method through a gel layer. The three-dimensional structure of the enzyme was determined by the molecular-replacement method at 0.99 Å resolution. The structural features are considered, and the structure of E. coli PNP is compared with the structures of the free enzyme and its complexes with purine base derivatives established earlier. A comparison of the environment of the purine base in the complex of PNP with formycin A and of the pyrimidine base in the complex of uridine phosphorylase with thymidine revealed the main structural features of the base-binding sites. Coordinates of the atomic model determined with high accuracy were deposited in the Protein Data Bank (PDB_ID: 4RJ2).

  10. Diastereoselective Synthesis of Glycosyl Phosphates by Using a Phosphorylase-Phosphatase Combination Catalyst.

    PubMed

    Wildberger, Patricia; Pfeiffer, Martin; Brecker, Lothar; Nidetzky, Bernd

    2015-12-21

    Sugar phosphates play an important role in metabolism and signaling, but also as constituents of macromolecular structures. Selective phosphorylation of sugars is chemically difficult, particularly at the anomeric center. We report phosphatase-catalyzed diastereoselective "anomeric" phosphorylation of various aldose substrates with α-D-glucose 1-phosphate, derived from phosphorylase-catalyzed conversion of sucrose and inorganic phosphate, as the phosphoryl donor. Simultaneous and sequential two-step transformations by the phosphorylase-phosphatase combination catalyst yielded glycosyl phosphates of defined anomeric configuration in yields of up to 70 % based on the phosphate applied to the reaction. An efficient enzyme-assisted purification of the glycosyl phosphate products from reaction mixtures was established.

  11. L-Enantiomers of Transition State Analogue Inhibitors Bound to Human Purine Nucleoside Phosphorylase

    SciTech Connect

    Rinaldo-Matthis,A.; Murkin, A.; Ramagopal, U.; Clinch, K.; Mee, S.; Evans, G.; Tyler, P.; Furneaux, R.; Almo, S.; Schramm, v.

    2008-01-01

    Human purine nucleoside phosphorylase (PNP) was crystallized with transition-state analogue inhibitors Immucillin-H and DADMe-Immucillin-H synthesized with ribosyl mimics of l-stereochemistry. The inhibitors demonstrate that major driving forces for tight binding of these analogues are the leaving group interaction and the cationic mimicry of the transition state, even though large geometric changes occur with d-Immucillins and l-Immucillins bound to human PNP.

  12. Crystallization and X-ray diffraction studies of cellobiose phosphorylase from Cellulomonas uda.

    PubMed

    Van Hoorebeke, Annelies; Stout, Jan; Kyndt, John; De Groeve, Manu; Dix, Ina; Desmet, Tom; Soetaert, Wim; Van Beeumen, Jozef; Savvides, Savvas N

    2010-03-01

    Disaccharide phosphorylases are able to catalyze both the synthesis and the breakdown of disaccharides and have thus emerged as attractive platforms for tailor-made sugar synthesis. Cellobiose phosphorylase from Cellulomonas uda (CPCuda) is an enzyme that belongs to glycoside hydrolase family 94 and catalyzes the reversible breakdown of cellobiose [beta-D-glucopyranosyl-(1,4)-D-glucopyranose] to alpha-D-glucose-1-phosphate and D-glucose. Crystals of ligand-free recombinant CPCuda and of its complexes with substrates and reaction products yielded complete X-ray diffraction data sets to high resolution using synchrotron radiation but suffered from significant variability in diffraction quality. In at least one case an intriguing space-group transition from a primitive monoclinic to a primitive orthorhombic lattice was observed during data collection. The structure of CPCuda was determined by maximum-likelihood molecular replacement, thus establishing a starting point for an investigation of the structural and mechanistic determinants of disaccharide phosphorylase activity.

  13. [Properties of sucrose phosphorylase from recombinant Escherichia coli and enzymatic synthesis of alpha-arbutin].

    PubMed

    Wan, Yuejia; Ma, Jiangfeng; Xu, Rong; He, Aiyong; Jiang, Min; Chen, Kequan; Jiang, Yin

    2012-12-01

    Sucrose phosphorylase (EC 2.4.1.7, Sucrose phosphorylase, SPase) can be produced by recombinant strain Escherichia coli Rosetta(DE3)/Pet-SPase. Crude enzyme was obtained from the cells by the high pressure disruption and centrifugation. Sucrose phosphorylase was purified by Ni-NTA affinity column chromatography and desalted by ultrafiltration. The specific enzyme activity was 1.1-fold higher than that of the crude enzyme, and recovery rate was 82.7%. The purified recombinant SPase had a band of 59 kDa on SDS-PAGE. Thermostability of the enzyme was shown at temperatures up to 37 degrees C, and pH stability between pH 6.0 and 6.7. The optimum temperature and pH were 37 degrees C and 6.7, respectively. The K(m) of SPase for sucrose was 7.3 mmol/L, and Vmax was 0.2 micromol/(min x mg). Besides, alpha-arbutin was synthesized from sucrose and hydroquinone by transglucosylation with recombinant SPase. The optimal conditions for synthesis of alpha-arbutin were 200 U/mL of recombinant SPase, 20% of sucrose, and 1.6% hydroquinone at pH 6-6.5 and 25 degrees C for 21 h. Under these conditions, alpha-arbutin was obtained with a 78.3% molar yield with respect to hydroquinone, and the concentration of alpha-arbutin was about 31 g/L.

  14. Glycogen phosphorylase is involved in stress endurance and biofilm formation in Azospirillum brasilense Sp7.

    PubMed

    Lerner, Anat; Castro-Sowinski, Susana; Lerner, Hadas; Okon, Yaacov; Burdman, Saul

    2009-11-01

    Here we report the identification of a glycogen phosphorylase (glgP) gene in the plant growth-promoting rhizobacterium Azospirillum brasilense, Sp7, and the characterization of a glgP marker exchange mutant of this strain. The glgP mutant showed a twofold reduction of glycogen phosphorylase activity and an increased glycogen accumulation as compared with wild-type Sp7, indicating that the identified gene indeed encodes a protein with glycogen phosphorylase activity. Interestingly, the glgP mutant had higher survival rates than the wild type after exposure to starvation, desiccation and osmotic pressure. The mutant was shown to be compromised in its biofilm formation ability. Analysis of the exopolysaccharide sugar composition of the glgP mutant revealed a decrease in the amount of glucose, accompanied by increases in rhamnose, fucose and ribose, as compared with the Sp7 exopolysaccharide. To the best of our knowledge, this is the first study that demonstrates GlgP activity in A. brasilense, and shows that glycogen accumulation may play an important role in the stress endurance of this bacterium.

  15. Structure analysis of archaeal AMP phosphorylase reveals two unique modes of dimerization.

    PubMed

    Nishitani, Yuichi; Aono, Riku; Nakamura, Akira; Sato, Takaaki; Atomi, Haruyuki; Imanaka, Tadayuki; Miki, Kunio

    2013-08-01

    AMP phosphorylase (AMPpase) catalyzes the initial reaction in a novel AMP metabolic pathway recently found in archaea, converting AMP and phosphate into adenine and ribose 1,5-bisphosphate. Gel-filtration chromatography revealed that AMPpase from Thermococcus kodakarensis (Tk-AMPpase) forms an exceptionally large macromolecular structure (>40-mers) in solution. To investigate its unique multimerization feature, we determined the first crystal structures of Tk-AMPpase, in the apo-form and in complex with substrates. Structures of two truncated forms of Tk-AMPpase (Tk-AMPpaseΔN84 and Tk-AMPpaseΔC10) clarified that this multimerization is achieved by two dimer interfaces within a single molecule: one by the central domain and the other by the C-terminal domain, which consists of an unexpected domain-swapping interaction. The N-terminal domain, characteristic of archaeal enzymes, is essential for enzymatic activity, participating in multimerization as well as domain closure of the active site upon substrate binding. Moreover, biochemical analysis demonstrated that the macromolecular assembly of Tk-AMPpase contributes to its high thermostability, essential for an enzyme from a hyperthermophile. Our findings unveil a unique archaeal nucleotide phosphorylase that is distinct in both function and structure from previously known members of the nucleoside phosphorylase II family.

  16. FR258900, a potential anti-hyperglycemic drug, binds at the allosteric site of glycogen phosphorylase

    PubMed Central

    Tiraidis, Costas; Alexacou, Kyra-Melinda; Zographos, Spyros E.; Leonidas, Demetres D.; Gimisis, Thanasis; Oikonomakos, Nikos G.

    2007-01-01

    FR258900 has been discovered as a novel inhibitor of human liver glycogen phosphorylase a and proved to suppress hepatic glycogen breakdown and reduce plasma glucose concentrations in diabetic mice models. To elucidate the mechanism of inhibition, we have determined the crystal structure of the cocrystallized rabbit muscle glycogen phosphorylase b–FR258900 complex and refined it to 2.2 Å resolution. The structure demonstrates that the inhibitor binds at the allosteric activator site, where the physiological activator AMP binds. The contacts from FR258900 to glycogen phosphorylase are dominated by nonpolar van der Waals interactions with Gln71, Gln72, Phe196, and Val45′ (from the symmetry-related subunit), and also by ionic interactions from the carboxylate groups to the three arginine residues (Arg242, Arg309, and Arg310) that form the allosteric phosphate-recognition subsite. The binding of FR258900 to the protein promotes conformational changes that stabilize an inactive T-state quaternary conformation of the enzyme. The ligand-binding mode is different from those of the potent phenoxy-phthalate and acyl urea inhibitors, previously described, illustrating the broad specificity of the allosteric site. PMID:17600143

  17. Compressional characteristics of four starches.

    PubMed

    Paronen, P; Juslin, M

    1983-10-01

    Compression data about barley, corn, potato and wheat starches were obtained by two methods: the ejected tablet method and the tablet-in-die-method. These data were analysed using the Heckel and the Cooper-Eaton equations. The Heckel equation appeared to be the more sensitive in distinguishing the various stages during the compression. Die filling and rearrangement processes for the starches were especially dependent on particle size and shape and thus on contact area between particles. Densification of large starch particles (potato starch) owed more to die filling and less to rearrangement. Densification of small particles (corn starch) was the reverse. Starch having a wide particle size distribution (wheat) or an irregular particle shape (barley) underwent a relatively small amount of densification as a result of die filling and a relatively great amount of densification because of rearrangement of particles during tableting. The tendency of the starches to total and pure plastic deformation was dependent on particle size, size distribution and particle shape. Corn starch was the most prone to plastic flow with only little elastic recovery. Potato starch also flowed plastically with ease. Barley and wheat starches were the more elastic. PMID:6139430

  18. Re-Evaluation of the Role of Starch in Gravitropic Sensing

    NASA Technical Reports Server (NTRS)

    Sack, Fred D.

    1998-01-01

    Plant organs grow toward or away from gravity as a way to orient those organs for optimizing growth. Starch has long been thought to be important in sensing the direction of the g-vector in gravitropism, but that hypothesis has also evoked controversy. We have previously shown that starch-deficient mutants of Arabidopsis (TC7) and Nicotiana (NS458) are impaired in their gravitropism. While this suggests that starch is not necessary for reduced gravitropism, it also indicates that the mass of the starch contributes to sensing when present and thus is necessary for full gravitropic sensitivity. The research supported by this grant focused on three related projects, (1) the effect of light on hypocotyl gravitropism in NS458, (2) the effects of root phototropism on measurements of gravitropic sensitivity, and (3) the effects of starch overproduction on sedimentation and gravitropism. Collectively, our results provide additional strong support for the importance of starch in gravitropic sensing. First, by accounting for negative phototropism in roots of two starchless mutants of Arabidopsis we have established that these mutants are much less sensitive to gravity than previously thought. This work also demonstrates the importance of designing experimental protocols that remove the influence of root phototropism on measuring root gravitropism. Second, light apparently promotes gravitropism in starch-deficient Nicotiana hypocotyls by increasing the trace amounts of starch in the plastids, by inducing limited plastid sedimentation and thus by presumably increasing the signal provided by plastid mass. And finally, we show that excess starch in Arabidopsis seedlings has little effect on gravitropic sensitivity implying that the sensing system is already saturated. However, in light-grown stems where this mutation results in starch accumulation and where the wild-type practically lacks starch in the sensing cells, the mutant is much more sensitive than the wild-type again

  19. Molecular and supra-molecular structure of waxy starches developed from cassava (Manihot esculenta Crantz).

    PubMed

    Rolland-Sabaté, Agnès; Sanchez, Teresa; Buléon, Alain; Colonna, Paul; Ceballos, Hernan; Zhao, Shan-Shan; Zhang, Peng; Dufour, Dominique

    2013-02-15

    The aim of this work was to characterize the amylopectin of low amylose content cassava starches obtained from transgenesis comparatively with a natural waxy cassava starch (WXN) discovered recently in CIAT (International Center for Tropical Agriculture). Macromolecular features, starch granule morphology, crystallinity and thermal properties of these starches were determined. M¯(w) of amylopectin from the transgenic varieties are lower than WXN. Branched and debranched chain distributions analyses revealed slight differences in the branching degree and structure of these amylopectins, principally on DP 6-9 and DP>37. For the first time, a deep structural characterization of a series of transgenic lines of waxy cassava was carried out and the link between structural features and the mutated gene expression approached. The transgenesis allows to silenced partially or totally the GBSSI, without changing deeply the starch granule ultrastructure and allows to produce clones with similar amylopectin as parental cassava clone.

  20. Interaction of Bacillus subtilis Polynucleotide Phosphorylase and RNase Y: STRUCTURAL MAPPING AND EFFECT ON mRNA TURNOVER.

    PubMed

    Salvo, Elizabeth; Alabi, Shanique; Liu, Bo; Schlessinger, Avner; Bechhofer, David H

    2016-03-25

    Polynucleotide phosphorylase (PNPase), a 3'-to-5' phosphorolytic exoribonuclease, is thought to be the primary enzyme responsible for turnover ofBacillus subtilismRNA. The role of PNPase inB. subtilismRNA decay has been analyzed previously by comparison of mRNA profiles in a wild-type strainversusa strain that is deleted forpnpA, the gene encoding PNPase. Recent studies have provided evidence for a degradosome-like complex inB. subtilisthat is built around the major decay-initiating endonuclease, RNase Y, and there is ample evidence for a strong interaction between PNPase and RNase Y. The role of the PNPase-RNase Y interaction in the exonucleolytic function of PNPase needs to be clarified. We sought to construct aB. subtilisstrain containing a catalytically active PNPase that could not interact with RNase Y. Mapping studies of the PNPase-RNase Y interaction were guided by a homology model ofB. subtilisPNPase based on the known structure of theEscherichia coliPNPase in complex with an RNase E peptide. Mutations inB. subtilisresidues predicted to be involved in RNase Y binding showed a loss of PNPase-RNase Y interaction. Two mRNAs whose decay is dependent on RNase Y and PNPase were examined in strains containing full-length PNPase that was either catalytically active but unable to interact with RNase Y, or catalytically inactive but able to interact with RNase Y. At least for these two mRNAs, disruption of the PNPase-RNase Y interaction did not appear to affect mRNA turnover.

  1. [Interaction of negative (CytT) and positive (cAMP-CRP) regulation in the promoter region of the uridine phosphorylase (udp) gene in Escherichia coli K-12].

    PubMed

    Mironov, A S; Nechaeva, G D; Sukhodolets, V V

    1989-03-01

    Interaction of negative (CytR) and positive (cAMP-CRP) control in the promoter region of the uridine phosphorylase (udp) gene of Escherichia coli has been studied by using udp-lac operon fusions in which the structural lacZ gene is expressed from the wild type promoter udpP+ or from mutant promoters udpP1 and udpP18. The specific activity of beta-galactosidase was examined in these fusions in cytR+ and cytR- backgrounds after introduction of specific mutations in crp locus, crp* and crp(a) altering interaction of CRP protein with catabolite-sensitive promoters. The data obtained using crp* mutation confirm the proposed model of the udp gene regulation, according to which CytR repressor protein interferes with CRP binding site in the promoter-operator region of the udp gene and thereby prevents the positive action of cAMP-CRP complex on the udp expression. Additional data in favor of this model were obtained using crp(a) mutation which most probably alters the structure of CRP protein in such a way that it exhibits more high affinity to the udp promoter, as compared to the CytR repressor protein. Indeed, taken by itself, the crp(a) mutation did not lead to any increase in the expression of udpP+-lac fusion under the conditions of cAMP limitation (on glucose-grown cells), in spite of whether or not the CytR repressor was present. However, when combined with the ptsG mutation or when cells were grown on succinate medium, complete constitutive expression of udpP+-lac fusion is observed, even in the presence of the cytR gene product. The effect of the crp(a) mutation was virtually the same in strains harboring udpP1-lac fusion. These data are in accordance with suggestion that udpP1 is a mutation in the site of the promoter-operator region that responds to the cytR gene product, while the corresponding binding site for CRP protein is still unaltered in this mutant. On the other hand, the crp(a) mutation causes only slight alteration in the expression of udpP18-lac

  2. Isolation, crystallization and preliminary crystallographic analysis of Salmonella typhimurium uridine phosphorylase crystallized with 2,2′-anhydrouridine

    SciTech Connect

    Timofeev, Vladimir I.; Lashkov, Alexander A.; Gabdoulkhakov, Azat G.; Pavlyuk, Bogdan Ph.; Kachalova, Galina S.; Betzel, Christian

    2007-10-01

    S. typhimurium uridine phosphorylase has been isolated and crystallized in the presence of ligand. Uridine phosphorylase (UPh; EC 2.4.2.3) is a member of the pyrimidine nucleoside phosphorylase family of enzymes which catalyzes the phosphorolytic cleavage of the C—N glycoside bond of uridine, with the formation of ribose 1-phosphate and uracil. This enzyme has been shown to be important in the activation and catabolism of fluoropyrimidines. Modulation of its enzymatic activity may affect the therapeutic efficacy of chemotherapeutic agents. The structural investigation of the bacterial uridine phosphorylases, both unliganded and complexed with substrate/product analogues and inhibitors, may help in understanding the catalytic mechanism of the phosphorolytic cleavage of uridine. Salmonella typhimurium uridine phosphorylase has been crystallized with 2,2′-anhydrouridine. X-ray diffraction data were collected to 2.15 Å. Preliminary analysis of the diffraction data indicates that the crystal belongs to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 88.52, b = 123.98, c = 133.52 Å. The solvent content is 45.51%, assuming the presence of one hexamer molecule per asymmetric unit.

  3. Alpha-glucan phosphorylase from Escherichia coli. Cloning of the gene, and purification and characterization of the protein.

    PubMed

    Yu, F; Jen, Y; Takeuchi, E; Inouye, M; Nakayama, H; Tagaya, M; Fukui, T

    1988-09-25

    By using a synthetic oligonucleotide probe identical to a part of the gene for the Escherichia coli major outer membrane lipoprotein, we have cloned a gene from E. coli chromosomal DNA. However, the cloned gene was not one of the lipoprotein genes. The amino acid sequence deduced from its nucleotide sequence shows extensive similarities instead to alpha-glucan phosphorylase (EC 2.4.1.1). The gene, glgP, is located immediately downstream from glgA, the gene for glycogen synthase. The glgP gene was inserted into pUC9 vector and expressed in the presence of the lac inducer. The gene product was purified to apparent homogeneity as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In all chromatographies, the protein was eluted accompanied by a low phosphorylase activity. The final preparation showed phosphorolytic activity to various alpha-glucans, although the specific activity was extremely low compared to other alpha-glucan phosphorylases under the standard assay conditions. Its enzymatic activity, however, increased almost linearly as the concentration of glucan increased, reaching a value comparable with those of other phosphorylases. The amino acid sequence deduced was compared with those of alpha-glucan phosphorylases from other sources. PMID:3047129

  4. Molecular evolution and functional divergence of soluble starch synthase genes in cassava (manihot esculenta crantz).

    PubMed

    Yang, Zefeng; Wang, Yifan; Xu, Shuhui; Xu, Chenwu; Yan, Changjie

    2013-01-01

    Soluble starch synthases (SSs) are major enzymes involved in starch biosynthesis in plants. Cassava starch has many remarkable characteristics, which should be influenced by the evolution of SS genes in this starchy root crop. In this work, we performed a comprehensive phylogenetic and evolutionary analysis of the soluble starch synthases in cassava. Genome-wide identification showed that there are 9 genes encoding soluble starch synthases in cassava. All of the soluble starch synthases encoded by these genes contain both Glyco_transf_5 and Glycos_transf_1 domains, and a correlation analysis showed evidence of coevolution between these 2 domains in cassava SS genes. The SS genes in land plants can be divided into 6 subfamilies that were formed before the origin of seed plants, and species-specific expansion has contributed to the evolution of this family in cassava. A functional divergence analysis for this family provided statistical evidence for shifted evolutionary rates between the subfamilies of land plant soluble starch synthases. Although the main selective pressure acting on land plant SS genes was purifying selection, our results also revealed that point mutation with positive selection contributed to the evolution of 2 SS genes in cassava. The remarkable cassava starch characteristics might be the result of both the duplication and adaptive selection of SS genes.

  5. Genetic dissection of floridean starch synthesis in the cytosol of the model dinoflagellate Crypthecodinium cohnii.

    PubMed

    Dauvillée, David; Deschamps, Philippe; Ral, Jean-Philippe; Plancke, Charlotte; Putaux, Jean-Luc; Devassine, Jimi; Durand-Terrasson, Amandine; Devin, Aline; Ball, Steven G

    2009-12-15

    Starch defines an insoluble semicrystalline form of storage polysaccharides restricted to Archaeplastida (red and green algae, land plants, and glaucophytes) and some secondary endosymbiosis derivatives of the latter. While green algae and land-plants store starch in plastids by using an ADP-glucose-based pathway related to that of cyanobacteria, red algae, glaucophytes, cryptophytes, dinoflagellates, and apicomplexa parasites store a similar type of polysaccharide named floridean starch in their cytosol or periplast. These organisms are suspected to store their floridean starch from UDP-glucose in a fashion similar to heterotrophic eukaryotes. However, experimental proof of this suspicion has never been produced. Dinoflagellates define an important group of both photoautotrophic and heterotrophic protists. We now report the selection and characterization of a low starch mutant of the heterotrophic dinoflagellate Crypthecodinium cohnii. We show that the sta1-1 mutation of C. cohnii leads to a modification of the UDP-glucose-specific soluble starch synthase activity that correlates with a decrease in starch content and an alteration of amylopectin structure. These experimental results validate the UDP-glucose-based pathway proposed for floridean starch synthesis.

  6. Brucite nanoplate reinforced starch bionanocomposites

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In this paper the mechanical reinforcement in a series of bionanocomposites films based on starch and nano-sized brucite, Mg(OH)2, was investigated. Brucite nanoplates with an aspect ratio of 9.25 were synthesized by wet precipitation and incorporated into starch matrices at different concentrations...

  7. Starch-filled polymer composites

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This report describes the development of degradable polymer composites that can be made at room temperature without special equipments. The developed composites are made from ethyl cyanoacrylate and starch. The polymer composites produced by this procedure contain 60 wt% of starch with compressive s...

  8. Starch Applications for Delivery Systems

    NASA Astrophysics Data System (ADS)

    Li, Jason

    2013-03-01

    Starch is one of the most abundant and economical renewable biopolymers in nature. Starch molecules are high molecular weight polymers of D-glucose linked by α-(1,4) and α-(1,6) glycosidic bonds, forming linear (amylose) and branched (amylopectin) structures. Octenyl succinic anhydride modified starches (OSA-starch) are designed by carefully choosing a proper starch source, path and degree of modification. This enables emulsion and micro-encapsulation delivery systems for oil based flavors, micronutrients, fragrance, and pharmaceutical actives. A large percentage of flavors are encapsulated by spray drying in today's industry due to its high throughput. However, spray drying encapsulation faces constant challenges with retention of volatile compounds, oxidation of sensitive compound, and manufacturing yield. Specialty OSA-starches were developed suitable for the complex dynamics in spray drying and to provide high encapsulation efficiency and high microcapsule quality. The OSA starch surface activity, low viscosity and film forming capability contribute to high volatile retention and low active oxidation. OSA starches exhibit superior performance, especially in high solids and high oil load encapsulations compared with other hydrocolloids. The submission is based on research and development of Ingredion

  9. Starch-Lignin Baked Foams

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Starch-kraft lignin foams were prepared by a baking process. Replacing up to 20% of the starch with lignin has no effect on foam density or overall morphology. At 10% replacement, lignin marginally increases water resistance and modulus of elasticity but decreases strain at maximum stress. At 20% re...

  10. Synthesis of tartaric acid analogues of FR258900 and their evaluation as glycogen phosphorylase inhibitors.

    PubMed

    Varga, Gergely; Docsa, Tibor; Gergely, Pál; Juhász, László; Somsák, László

    2013-03-15

    Di-O-cinnamoylated, -p-coumaroylated, and -feruloylated d-, l- and meso-tartaric acids were synthesized as analogues of the natural product FR258900, a glycogen phosphorylase (GP) inhibitor with in vivo antihyperglycaemic activity. The new compounds inhibited rabbit muscle GP in the low micromolar range, and bound to the allosteric site of the enzyme. The best inhibitor was 2,3-di-O-feruloyl meso-tartaric acid and had Ki values of 2.0μM against AMP (competitive) and 3.36μM against glucose-1-phosphate (non-competitive).

  11. Preparation, proteolysis and reversible oxidation of highly purified Azotobacter vinelandii polynucleotide phosphorylase

    PubMed Central

    Gajda, A. T.; de Behrens, G. Zaror; Fitt, P. S.

    1970-01-01

    1. A new method has been developed for the preparation in good yield of highly purified Azotobacter vinelandii polynucleotide phosphorylase in its reduced form. 2. Aging or digestion with trypsin causes the enzyme to develop a primer requirement that is not eliminated by β-mercaptoethanol. 3. The development of a primer requirement is accompanied by marked changes of the electrophoretic mobility of the enzyme in polyacrylamide gels. 4. The enzyme is inactivated by aerial oxidation or thiol-specific reagents. The lost activity is restored by β-mercaptoethanol, but not by oligonucleotide primers. PMID:5495150

  12. Crystallographic and docking studies of purine nucleoside phosphorylase from Mycobacterium tuberculosis.

    PubMed

    Ducati, Rodrigo G; Basso, Luiz A; Santos, Diógenes S; de Azevedo, Walter F

    2010-07-01

    This work describes for the first time the structure of purine nucleoside phosphorylase from Mycobacterium tuberculosis (MtPNP) in complex with sulfate and its natural substrate, 2'-deoxyguanosine, and its application to virtual screening. We report docking studies of a set of molecules against this structure. Application of polynomial empirical scoring function was able to rank docking solutions with good predicting power which opens the possibility to apply this new criterion to analyze docking solutions and screen small-molecule databases for new chemical entities to inhibit MtPNP.

  13. Compositions and methods involving methyladenosine phosphorylase in the diagnosis and treatment of proliferative disorders

    DOEpatents

    Olopade, Olufunmilayo I.

    2007-03-20

    Disclosed are novel nucleic acid and peptide compositions comprising methylthioadenosine phosphorylase (MTAP) and methods of use for MTAP amino acid sequences and DNA segments comprising MTAP in the diagnosis of human cancers and development of MTAP-specific antibodies. Also disclosed are methods for the diagnosis and treatment of tumors and other proliferative cell disorders, and identification of tumor suppressor genes and gene products from the human 9p21-p22 chromosome region. Such methods are useful in the diagnosis of multiple tumor types such as bladder cancer, lung cancer, breast cancer, pancreatic cancer, brain tumors, lymphomas, gliomas, melanomas, and leukemias.

  14. Methylthioadenosine phosphorylase compositions and methods of use in the diagnosis and treatment of proliferative disorders

    DOEpatents

    Olopade, Olufunmilayo I.

    2005-03-22

    Disclosed are novel nucleic acid and peptide compositions comprising methythlioadenosine phosphorylase (MTAP) and methods of use for MTAP amino acid sequences and DNA segments comprising MTAP in the diagnosis of human cancers and development of MTAP-specific antibodies. Also disclosed are methods for the diagnosis and treatment of tumors and other proliferative cell disorders, and idenification tumor suppressor genes and gene products from the human 9p21-p22 chromosome region. Such methods are useful in the diagnosis of multiple tumor types such as bladder cancer, lung cancer, breast cancer, pancreatic cancer, brain tumors, lymphomas, gliomas, melanomas, and leukemias.

  15. Starches, Sugars and Obesity

    PubMed Central

    Aller, Erik E. J. G.; Abete, Itziar; Astrup, Arne; Martinez, J. Alfredo; van Baak, Marleen A.

    2011-01-01

    The rising prevalence of obesity, not only in adults but also in children and adolescents, is one of the most important public health problems in developed and developing countries. As one possible way to tackle obesity, a great interest has been stimulated in understanding the relationship between different types of dietary carbohydrate and appetite regulation, body weight and body composition. The present article reviews the conclusions from recent reviews and meta-analyses on the effects of different starches and sugars on body weight management and metabolic disturbances, and provides an update of the most recent studies on this topic. From the literature reviewed in this paper, potential beneficial effects of intake of starchy foods, especially those containing slowly-digestible and resistant starches, and potential detrimental effects of high intakes of fructose become apparent. This supports the intake of whole grains, legumes and vegetables, which contain more appropriate sources of carbohydrates associated with reduced risk of cardiovascular and other chronic diseases, rather than foods rich in sugars, especially in the form of sugar-sweetened beverages. PMID:22254101

  16. Starch synthesis in potato tubers is regulated by post-translational redox modification of ADP-glucose pyrophosphorylase: a novel regulatory mechanism linking starch synthesis to the sucrose supply.

    PubMed

    Tiessen, Axel; Hendriks, Janneke H M; Stitt, Mark; Branscheid, Anja; Gibon, Yves; Farré, Eva M; Geigenberger, Peter

    2002-09-01

    Transcriptional and allosteric regulation of ADP-Glc pyrophosphorylase (AGPase) plays a major role in the regulation of starch synthesis. Analysis of the response after detachment of growing potato tubers from the mother plant revealed that this concept requires extension. Starch synthesis was inhibited within 24 h of tuber detachment, even though the catalytic subunit of AGPase (AGPB) and overall AGPase activity remained high, the substrates ATP and Glc-1-P increased, and the glycerate-3-phosphate/inorganic orthophosphate (the allosteric activator and inhibitor, respectively) ratio increased. This inhibition was abolished in transformants in which a bacterial AGPase replaced the potato AGPase. Measurements of the subcellular levels of each metabolite between Suc and starch established AGPase as the only step whose substrates increase and mass action ratio decreases after detachment of wild-type tubers. Separation of extracts on nonreducing SDS gels revealed that AGPB is present as a mixture of monomers and dimers in growing tubers and becomes dimerized completely in detached tubers. Dimerization led to inactivation of the enzyme as a result of a marked decrease of the substrate affinity and sensitivity to allosteric effectors. Dimerization could be reversed and AGPase reactivated in vitro by incubating extracts with DTT. Incubation of tuber slices with DTT or high Suc levels reduced dimerization, increased AGPase activation, and stimulated starch synthesis in vivo. In intact tubers, the Suc content correlated strongly with AGPase activation across a range of treatments, including tuber detachment, aging of the mother plant, heterologous overexpression of Suc phosphorylase, and antisense inhibition of endogenous AGPase activity. Furthermore, activation of AGPase resulted in a stimulation of starch synthesis and decreased levels of glycolytic intermediates.

  17. Purification and characterization of purine nucleoside phosphorylase from developing embryos of Hyalomma dromedarii.

    PubMed

    Kamel, M Y; Fahmy, A S; Ghazy, A H; Mohamed, M A

    1991-04-01

    Purine nucleoside phosphorylase from Hyalomma dromedarii, the camel tick, was purified to apparent homogeneity. A molecular weight of 56,000 - 58,000 was estimated for both the native and denatured enzyme, suggesting that the enzyme is monomeric. Unlike purine nucleoside phosphorylase preparations from other tissues, the H. dromedarii enzyme was unstable in the presence of beta-mercaptoethanol. The enzyme had a sharp pH optimum at pH 6.5. It catalyzed the phosphorolysis and arsenolysis of ribo- and deoxyribo-nucleosides of hypoxanthine and guanine, but not of adenine or pyrimidine nucleosides. The Km values of the enzyme at the optimal pH for inosine, deoxyinosine, guanosine, and deoxyguanosine were 0.31, 0.67, 0.55, and 0.33 mM, respectively. Inactivation and kinetic studies suggested that histidine and cysteine residues were essential for activity. The pKa values determined for catalytic ionizable groups were 6-7 and 8-9. The enzyme was completely inactivated by thiol reagents and reactivated by excess beta-mercaptoethanol. The enzyme was also susceptible to pH-dependent photooxidation in the presence of methylene blue, implicating histidine. Initial velocity studies showed an intersecting pattern of double-reciprocal plots of the data, consistent with a sequential mechanism. PMID:1905141

  18. Structural determinants of the 5'-methylthioinosine specificity of Plasmodium purine nucleoside phosphorylase.

    PubMed

    Donaldson, Teraya M; Ting, Li-Min; Zhan, Chenyang; Shi, Wuxian; Zheng, Renjian; Almo, Steven C; Kim, Kami

    2014-01-01

    Plasmodium parasites rely upon purine salvage for survival. Plasmodium purine nucleoside phosphorylase is part of the streamlined Plasmodium purine salvage pathway that leads to the phosphorylysis of both purines and 5'-methylthiopurines, byproducts of polyamine synthesis. We have explored structural features in Plasmodium falciparum purine nucleoside phosphorylase (PfPNP) that affect efficiency of catalysis as well as those that make it suitable for dual specificity. We used site directed mutagenesis to identify residues critical for PfPNP catalytic activity as well as critical residues within a hydrophobic pocket required for accommodation of the 5'-methylthio group. Kinetic analysis data shows that several mutants had disrupted binding of the 5'-methylthio group while retaining activity for inosine. A triple PfPNP mutant that mimics Toxoplasma gondii PNP had significant loss of 5'-methylthio activity with retention of inosine activity. Crystallographic investigation of the triple mutant PfPNP with Tyr160Phe, Val66Ile, andVal73Ile in complex with the transition state inhibitor immucillin H reveals fewer hydrogen bond interactions for the inhibitor in the hydrophobic pocket. PMID:24416224

  19. Enzymatic Properties and Substrate Specificity of the Trehalose Phosphorylase from Caldanaerobacter subterraneus▿

    PubMed Central

    Van der Borght, Jef; Chen, Chao; Hoflack, Lieve; Van Renterghem, Lucas; Desmet, Tom; Soetaert, Wim

    2011-01-01

    A putative glycoside phosphorylase from Caldanaerobacter subterraneus subsp. pacificus was recombinantly expressed in Escherichia coli, after codon optimization and chemical synthesis of the encoding gene. The enzyme was purified by His tag chromatography and was found to be specifically active toward trehalose, with an optimal temperature of 80°C. In addition, no loss of activity could be detected after 1 h of incubation at 65°C, which means that it is the most stable trehalose phosphorylase reported so far. The substrate specificity was investigated in detail by measuring the relative activity on a range of alternative acceptors, applied in the reverse synthetic reaction, and determining the kinetic parameters for the best acceptors. These results were rationalized based on the enzyme-substrate interactions observed in a homology model with a docked ligand. The specificity for the orientation of the acceptor's hydroxyl groups was found to decrease in the following order: C-3 > C-2 > C-4. This results in a particularly high activity on the monosaccharides d-fucose, d-xylose, l-arabinose, and d-galactose, as well as on l-fucose. However, determination of the kinetic parameters revealed that these acceptors bind less tightly in the active site than the natural acceptor d-glucose, resulting in drastically increased Km values. Nevertheless, the enzyme's high thermostability and broad acceptor specificity make it a valuable candidate for industrial disaccharide synthesis. PMID:21803886

  20. Structural determinants of the 5'-methylthioinosine specificity of Plasmodium purine nucleoside phosphorylase.

    PubMed

    Donaldson, Teraya M; Ting, Li-Min; Zhan, Chenyang; Shi, Wuxian; Zheng, Renjian; Almo, Steven C; Kim, Kami

    2014-01-01

    Plasmodium parasites rely upon purine salvage for survival. Plasmodium purine nucleoside phosphorylase is part of the streamlined Plasmodium purine salvage pathway that leads to the phosphorylysis of both purines and 5'-methylthiopurines, byproducts of polyamine synthesis. We have explored structural features in Plasmodium falciparum purine nucleoside phosphorylase (PfPNP) that affect efficiency of catalysis as well as those that make it suitable for dual specificity. We used site directed mutagenesis to identify residues critical for PfPNP catalytic activity as well as critical residues within a hydrophobic pocket required for accommodation of the 5'-methylthio group. Kinetic analysis data shows that several mutants had disrupted binding of the 5'-methylthio group while retaining activity for inosine. A triple PfPNP mutant that mimics Toxoplasma gondii PNP had significant loss of 5'-methylthio activity with retention of inosine activity. Crystallographic investigation of the triple mutant PfPNP with Tyr160Phe, Val66Ile, andVal73Ile in complex with the transition state inhibitor immucillin H reveals fewer hydrogen bond interactions for the inhibitor in the hydrophobic pocket.

  1. Structural basis for the mechanism of inhibition of uridine phosphorylase from Salmonella typhimurium

    SciTech Connect

    Lashkov, A. A.; Zhukhlistova, N. E.; Sotnichenko, S. E.; Gabdulkhakov, A. G.; Mikhailov, A. M.

    2010-01-15

    The three-dimensional structures of three complexes of Salmonella typhimurium uridine phosphorylase with the inhibitor 2,2'-anhydrouridine, the substrate PO{sub 4}, and with both the inhibitor 2,2'-anhydrouridine and the substrate PO{sub 4} (a binary complex) were studied in detail by X-ray diffraction. The structures of the complexes were refined at 2.38, 1.5, and 1.75 A resolution, respectively. Changes in the three-dimensional structure of the subunits in different crystal structures are considered depending on the presence or absence of the inhibitor molecule and (or) the phosphate ion in the active site of the enzyme. The presence of the phosphate ion in the phosphate-binding site was found to substantially change the orientations of the side chains of the amino-acid residues Arg30, Arg91, and Arg48 coordinated to this ion. A comparison showed that the highly flexible loop L9 is unstable. The atomic coordinates of the refined structures of the complexes and the corresponding structure factors were deposited in the Protein Data Bank (their PDB ID codes are 3DD0 and 3C74). The experimental data on the spatial reorganization of the active site caused by changes in its functional state from the unligated to the completely inhibited state suggest the structural basis for the mechanism of inhibition of Salmonella typhimurium uridine phosphorylase.

  2. Adsorption and enzyme activity of sucrose phosphorylase on lipid Langmuir and Langmuir-Blodgett films.

    PubMed

    Rocha, Jefferson Muniz; Caseli, Luciano

    2014-04-01

    The production of bioelectronic devices, including biosensors, can be conducted using enzymes immobilized in ultrathin solid films, for which preserving the enzymatic catalytic activity is crucial for optimal performance. In this sense, nanostructured films that allow for control over molecular architectures are of interest. In this paper, we investigate the adsorption of sucrose phosphorylase onto Langmuir monolayers of the phospholipid dimyristoylphosphatidic acid, which caused the surface pressure isotherms to expand. With polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS), the amide bands from the enzyme could be identified, with the C-N and C=O dipole moments lying parallel to the air-water interface. Structuring of the enzyme into an α-helix was noted, and this structure was preserved when the mixed enzyme-phospholipid monolayer was transferred in the form of a Langmuir-Blodgett (LB) film. The latter was demonstrated with measurements of the catalytic activity of sucrose phosphorylase, which presented the highest enzyme activity for multilayer LB film. The approach presented in this study not only allows for optimized catalytic activity toward sucrose but also permits to explain why certain film architectures exhibit superior performance.

  3. The experimental type 2 diabetes therapy glycogen phosphorylase inhibition can impair aerobic muscle function during prolonged contraction.

    PubMed

    Baker, David J; Greenhaff, Paul L; MacInnes, Alan; Timmons, James A

    2006-06-01

    Glycogen phosphorylase inhibition represents a promising strategy to suppress inappropriate hepatic glucose output, while muscle glycogen is a major source of fuel during contraction. Glycogen phosphorylase inhibitors (GPi) currently being investigated for the treatment of type 2 diabetes do not demonstrate hepatic versus muscle glycogen phosphorylase isoform selectivity and may therefore impair patient aerobic exercise capabilities. Skeletal muscle energy metabolism and function are not impaired by GPi during high-intensity contraction in rat skeletal muscle; however, it is unknown whether glycogen phosphorylase inhibitors would impair function during prolonged lower-intensity contraction. Utilizing a novel red cell-perfused rodent gastrocnemius-plantaris-soleus system, muscle was pretreated for 60 min with either 3 micromol/l free drug GPi (n=8) or vehicle control (n=7). During 60 min of aerobic contraction, GPi treatment resulted in approximately 35% greater fatigue. Muscle glycogen phosphorylase a form (P<0.01) and maximal activity (P<0.01) were reduced in the GPi group, and postcontraction glycogen (121.8 +/- 16.1 vs. 168.3 +/- 8.5 mmol/kg dry muscle, P<0.05) was greater. Furthermore, lower muscle lactate efflux and glucose uptake (P<0.01), yet higher muscle Vo(2), support the conclusion that carbohydrate utilization was impaired during contraction. Our data provide new confirmation that muscle glycogen plays an essential role during submaximal contraction. Given the critical role of exercise prescription in the treatment of type 2 diabetes, it will be important to monitor endurance capacity during the clinical evaluation of nonselective GPi. Alternatively, greater effort should be devoted toward the discovery of hepatic-selective GPi, hepatic-specific drug delivery strategies, and/or alternative strategies for controlling excess hepatic glucose production in type 2 diabetes.

  4. Deficiency of maize starch-branching enzyme i results in altered starch fine structure, decreased digestibility and reduced coleoptile growth during germination

    PubMed Central

    2011-01-01

    Background Two distinct starch branching enzyme (SBE) isoforms predate the divergence of monocots and dicots and have been conserved in plants since then. This strongly suggests that both SBEI and SBEII provide unique selective advantages to plants. However, no phenotype for the SBEI mutation, sbe1a, had been previously observed. To explore this incongruity the objective of the present work was to characterize functional and molecular phenotypes of both sbe1a and wild-type (Wt) in the W64A maize inbred line. Results Endosperm starch granules from the sbe1a mutant were more resistant to digestion by pancreatic α-amylase, and the sbe1a mutant starch had an altered branching pattern for amylopectin and amylose. When kernels were germinated, the sbe1a mutant was associated with shorter coleoptile length and higher residual starch content, suggesting that less efficient starch utilization may have impaired growth during germination. Conclusions The present report documents for the first time a molecular phenotype due to the absence of SBEI, and suggests strongly that it is associated with altered physiological function of the starch in vivo. We believe that these results provide a plausible rationale for the conservation of SBEI in plants in both monocots and dicots, as greater seedling vigor would provide an important survival advantage when resources are limited. PMID:21599988

  5. Impact of Waxy, Partial Waxy, and Wildtype Wheat Starch Fraction Properties on Hearth Bread Characteristics

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Thirteen different wheat (Triticum aestivum L.)cultivars were selected to represent GBSS mutations: three each of wildtype, axnull, and bxnull, and two each of 2xnull and waxy. Starch and A- and B-granules were purified from wheat flour. Hearth bread loaves were produced from the flours using a smal...

  6. Kernel Composition, Starch Structure, and Enzyme Digestibility of Opaque-2 Maize and Quality Protein Maize

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Objectives of this study were to understand how opaque-2 (o2) mutation and quality protein maize (QPM) affect maize kernel composition and starch structure, property, and enzyme digestibility. Kernels of o2 maize contained less protein (9.6−12.5%) than those of the wild-type (WT) counterparts (12...

  7. Starch-degrading polysaccharide monooxygenases.

    PubMed

    Vu, Van V; Marletta, Michael A

    2016-07-01

    Polysaccharide degradation by hydrolytic enzymes glycoside hydrolases (GHs) is well known. More recently, polysaccharide monooxygenases (PMOs, also known as lytic PMOs or LPMOs) were found to oxidatively degrade various polysaccharides via a copper-dependent hydroxylation. PMOs were previously thought to be either GHs or carbohydrate binding modules (CBMs), and have been re-classified in carbohydrate active enzymes (CAZY) database as auxiliary activity (AA) families. These enzymes include cellulose-active fungal PMOs (AA9, formerly GH61), chitin- and cellulose-active bacterial PMOs (AA10, formerly CBM33), and chitin-active fungal PMOs (AA11). These PMOs significantly boost the activity of GHs under industrially relevant conditions, and thus have great potential in the biomass-based biofuel industry. PMOs that act on starch are the latest PMOs discovered (AA13), which has expanded our perspectives in PMOs studies and starch degradation. Starch-active PMOs have many common structural features and biochemical properties of the PMO superfamily, yet differ from other PMO families in several important aspects. These differences likely correlate, at least in part, to the differences in primary and higher order structures of starch and cellulose, and chitin. In this review we will discuss the discovery, structural features, biochemical and biophysical properties, and possible biological functions of starch-active PMOs, as well as their potential application in the biofuel, food, and other starch-based industries. Important questions regarding various aspects of starch-active PMOs and possible economical driving force for their future studies will also be highlighted. PMID:27170366

  8. Enzymatic Glycosylation of Phenolic Antioxidants: Phosphorylase-Mediated Synthesis and Characterization.

    PubMed

    De Winter, Karel; Dewitte, Griet; Dirks-Hofmeister, Mareike E; De Laet, Sylvie; Pelantová, Helena; Křen, Vladimír; Desmet, Tom

    2015-11-25

    Although numerous biologically active molecules exist as glycosides in nature, information on the activity, stability, and solubility of glycosylated antioxidants is rather limited to date. In this work, a wide variety of antioxidants were glycosylated using different phosphorylase enzymes. The resulting antioxidant library, containing α/β-glucosides, different regioisomers, cellobiosides, and cellotriosides, was then characterized. Glycosylation was found to significantly increase the solubility and stability of all evaluated compounds. Despite decreased radical-scavenging abilities, most glycosides were identified to be potent antioxidants, outperforming the commonly used 2,6-bis(1,1-dimethylethyl)-4-methylphenol (BHT). Moreover, the point of attachment, the anomeric configuration, and the glycosidic chain length were found to influence the properties of these phenolic glycosides.

  9. Enzymatic Glycosylation of Phenolic Antioxidants: Phosphorylase-Mediated Synthesis and Characterization.

    PubMed

    De Winter, Karel; Dewitte, Griet; Dirks-Hofmeister, Mareike E; De Laet, Sylvie; Pelantová, Helena; Křen, Vladimír; Desmet, Tom

    2015-11-25

    Although numerous biologically active molecules exist as glycosides in nature, information on the activity, stability, and solubility of glycosylated antioxidants is rather limited to date. In this work, a wide variety of antioxidants were glycosylated using different phosphorylase enzymes. The resulting antioxidant library, containing α/β-glucosides, different regioisomers, cellobiosides, and cellotriosides, was then characterized. Glycosylation was found to significantly increase the solubility and stability of all evaluated compounds. Despite decreased radical-scavenging abilities, most glycosides were identified to be potent antioxidants, outperforming the commonly used 2,6-bis(1,1-dimethylethyl)-4-methylphenol (BHT). Moreover, the point of attachment, the anomeric configuration, and the glycosidic chain length were found to influence the properties of these phenolic glycosides. PMID:26540621

  10. The ligand binding mechanism to purine nucleoside phosphorylase elucidated via molecular dynamics and machine learning

    PubMed Central

    Decherchi, Sergio; Berteotti, Anna; Bottegoni, Giovanni; Rocchia, Walter; Cavalli, Andrea

    2015-01-01

    The study of biomolecular interactions between a drug and its biological target is of paramount importance for the design of novel bioactive compounds. In this paper, we report on the use of molecular dynamics (MD) simulations and machine learning to study the binding mechanism of a transition state analogue (DADMe–immucillin-H) to the purine nucleoside phosphorylase (PNP) enzyme. Microsecond-long MD simulations allow us to observe several binding events, following different dynamical routes and reaching diverse binding configurations. These simulations are used to estimate kinetic and thermodynamic quantities, such as kon and binding free energy, obtaining a good agreement with available experimental data. In addition, we advance a hypothesis for the slow-onset inhibition mechanism of DADMe–immucillin-H against PNP. Combining extensive MD simulations with machine learning algorithms could therefore be a fruitful approach for capturing key aspects of drug–target recognition and binding. PMID:25625196

  11. Evaluation of capillary chromatographic supports for immobilized human purine nucleoside phosphorylase in frontal affinity chromatography studies.

    PubMed

    de Moraes, Marcela Cristina; Temporini, Caterina; Calleri, Enrica; Bruni, Giovanna; Ducati, Rodrigo Gay; Santos, Diógenes Santiago; Cardoso, Carmen Lucia; Cass, Quezia Bezerra; Massolini, Gabriella

    2014-04-18

    The aim of this work was to optimize the preparation of a capillary human purine nucleoside phosphorylase (HsPNP) immobilized enzyme reactor (IMER) for characterization and affinity screening studies of new inhibitors by frontal affinity chromatography coupled to mass spectrometry (FAC-MS). For this purpose two monolithic supports, a Chromolith Speed Rod (0.1mm I.D.×5cm) and a methacrylate-based monolithic epoxy polymeric capillary column (0.25mm I.D.×5cm) with epoxy reactive groups were considered and compared to an IMER previously developed using an open fused silica capillary. Each HsPNP-IMER was characterized in terms of catalytic activity using Inosine as standard substrate. Furthermore, they were also explored for affinity ranking experiments. Kd determination was carried out with the based fused silica HsPNP-IMER and the results are herein discussed.

  12. Natural flavonoids as antidiabetic agents. The binding of gallic and ellagic acids to glycogen phosphorylase b.

    PubMed

    Kyriakis, Efthimios; Stravodimos, George A; Kantsadi, Anastassia L; Chatzileontiadou, Demetra S M; Skamnaki, Vassiliki T; Leonidas, Demetres D

    2015-07-01

    We present a study on the binding of gallic acid and its dimer ellagic acid to glycogen phosphorylase (GP). Ellagic acid is a potent inhibitor with Kis of 13.4 and 7.5 μM, in contrast to gallic acid which displays Kis of 1.7 and 3.9 mM for GPb and GPa, respectively. Both compounds are competitive inhibitors with respect to the substrate, glucose-1-phoshate, and non-competitive to the allosteric activator, AMP. However, only ellagic acid functions with glucose in a strongly synergistic mode. The crystal structures of the GPb-gallic acid and GPb-ellagic acid complexes were determined at high resolution, revealing that both ligands bind to the inhibitor binding site of the enzyme and highlight the structural basis for the significant difference in their inhibitory potency.

  13. Structure of cellobiose phosphorylase from Clostridium thermocellum in complex with phosphate

    PubMed Central

    Bianchetti, Christopher M.; Elsen, Nathaniel L.; Fox, Brian G.; Phillips, George N.

    2011-01-01

    Clostridium thermocellum is a cellulosome-producing bacterium that is able to efficiently degrade and utilize cellulose as a sole carbon source. Cellobiose phosphorylase (CBP) plays a critical role in cellulose degradation by catalyzing the reversible phosphate-dependent hydrolysis of cellobiose, the major product of cellulose degradation, into α-d-glucose 1-phosphate and d-glucose. CBP from C. thermocellum is a modular enzyme composed of four domains [N-­terminal domain, helical linker, (α/α)6-barrel domain and C-terminal domain] and is a member of glycoside hydrolase family 94. The 2.4 Å resolution X-ray crystal structure of C. thermocellum CBP reveals the residues involved in coordinating the catalytic phosphate as well as the residues that are likely to be involved in substrate binding and discrimination. PMID:22102229

  14. Human polynucleotide phosphorylase (hPNPase old-35): an RNA degradation enzyme with pleiotrophic biological effects.

    PubMed

    Sarkar, Devanand; Fisher, Paul B

    2006-05-01

    Identification of small inhibitory RNAs and microRNA established that regulation of RNA metabolism plays an essential role in controlling intracellular biochemical processes. Interferons induce a number of RNA degradation enzymes involved in innate immunity by degrading viral RNAs. We cloned human polynucleotide phosphorylase (hPNPase(old-35)), a type I interferon-inducible 3'-5' exoribonuclease, as a transcript induced during terminal differentiation and senescence, two physiological processes marked by irreversible growth arrest. Our studies in the last four years show that hPNPase(old-35) plays an essential role in mediating IFN-mediated growth inhibition and its upregulation might mediate chronic inflammatory pathological processes during aging. The present review recaps these findings and provides a framework for the future understanding of the versatile functions of this interesting molecule. PMID:16687933

  15. Nicotinamide riboside and nicotinic acid riboside salvage in fungi and mammals. Quantitative basis for Urh1 and purine nucleoside phosphorylase function in NAD+ metabolism.

    PubMed

    Belenky, Peter; Christensen, Kathryn C; Gazzaniga, Francesca; Pletnev, Alexandre A; Brenner, Charles

    2009-01-01

    NAD+ is a co-enzyme for hydride transfer enzymes and an essential substrate of ADP-ribose transfer enzymes and sirtuins, the type III protein lysine deacetylases related to yeast Sir2. Supplementation of yeast cells with nicotinamide riboside extends replicative lifespan and increases Sir2-dependent gene silencing by virtue of increasing net NAD+ synthesis. Nicotinamide riboside elevates NAD+ levels via the nicotinamide riboside kinase pathway and by a pathway initiated by splitting the nucleoside into a nicotinamide base followed by nicotinamide salvage. Genetic evidence has established that uridine hydrolase, purine nucleoside phosphorylase, and methylthioadenosine phosphorylase are required for Nrk-independent utilization of nicotinamide riboside in yeast. Here we show that mammalian purine nucleoside phosphorylase but not methylthioadenosine phosphorylase is responsible for mammalian nicotinamide riboside kinase-independent nicotinamide riboside utilization. We demonstrate that so-called uridine hydrolase is 100-fold more active as a nicotinamide riboside hydrolase than as a uridine hydrolase and that uridine hydrolase and mammalian purine nucleoside phosphorylase cleave nicotinic acid riboside, whereas the yeast phosphorylase has little activity on nicotinic acid riboside. Finally, we show that yeast nicotinic acid riboside utilization largely depends on uridine hydrolase and nicotinamide riboside kinase and that nicotinic acid riboside bioavailability is increased by ester modification. PMID:19001417

  16. Elevated thymidine phosphorylase activity in psoriatic lesions accounts for the apparent presence of an epidermal growth inhibitor, but is not in itself growth inhibitory

    SciTech Connect

    Hammerberg, C.; Fisher, G.J.; Voorhees, J.J.; Cooper, K.D. )

    1991-08-01

    An apparent tissue-specific growth inhibitor, or chalone, obtained from psoriatic lesions was tentatively identified in the 100-kDa fraction based upon inhibition of DNA synthesis, as measured by (3H)-thymidine uptake by a squamous cell carcinoma cell line, SCC 38. This fraction, however, failed to inhibit SCC 38 cell growth when assessed directly in a neutral red uptake assay. Characterization of the inhibitor of (3H)-thymidine uptake revealed it to have biochemical properties identical to thymidine phosphorylase: (1) molecular weight close to 100 kDa, (2) isoelectric point of 4.2, and (3) thymidine phosphorylase enzyme activity. Thus, the authors conclude that its ability to inhibit (3H)-thymidine uptake was due to thymidine catabolism rather than inhibition of DNA synthesis or growth inhibition. Examination of thymidine phosphorylase activity in keratome biopsies from psoriatic and normal skin demonstrated a twentyfold increase in activity in psoriatic lesions relative to non-lesional or normal skin. This increase in metabolism of thymidine was due to thymidine phosphorylase rather than uridine phosphorylase activity. The correlation between increased thymidine phosphorylase activity and increased keratinocyte proliferation in vitro (cultured) and in vivo (psoriasis), suggests that this enzyme may play a critical role in providing the thymidine necessary for keratinocyte proliferation.

  17. Rheological properties of reactive extrusion modified waxy starch and waxy starch-polyacrylamide copolymer gels

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The rheological properties of modified waxy starch and waxy starch-polyacrylamide graft copolymers prepared by reactive extrusion were investigated. Both materials can absorb huge amount of water and form gels. The modified waxy starch and waxy starch-polyacrylamide graft copolymer gels all exhibite...

  18. Comparison of Cationic and Unmodified Starches in Reactive Extrusion of Starch-Polyacrylamide Graft Copolymers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Graft copolymers of starch and polyacrylamide (PAAm) were prepared using reactive extrusion in a corotating twin screw extruder. The effect of cationic starch modification was examined using unmodified and cationic dent starch (approximately 23% amylose) and waxy maize starch (approximately 2% amyl...

  19. Isolation and partial characterization of banana starches.

    PubMed

    Bello-Pérez, L A; Agama-Acevedo, E; Sánchez-Hernández, L; Paredes-López, O

    1999-03-01

    Two varieties of banana green fruit growing in Guerrero, Mexico, were used for starch isolation. Chemical analysis and physicochemical and functional properties were studied in these starches. The "macho" variety presented higher starch yield than "criollo". In general, chemical compositions in both starches were similar, except in ash content, where the "criollo" variety showed a lower value than "macho". The results of freeze-thaw stability suggested that banana starches cannot be used in frozen products. Both starches presented similar water retention capacity values that increased when temperature increased. Solubility profiles showed that at low temperature "criollo" had lower solubility than "macho", but at higher temperature an inverse behavior was evident; also the solubility increased when temperature increased. Behavior similar to that for solubility was obtained in the swelling test. The banana starch studies indicate the "macho" and "criollo" varieties have different starch structures as evidenced by viscosity.

  20. Quality of Spelt Wheat and its Starch

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Flours from 5 spelt cultivars grown over 3 years were evaluated as to their bread baking quality and isolated starch properties. The starch properties included amylose contents, gelatinization temperatures (differential scanning calorimetry), granule size distributions and pasting properties. Mill...

  1. Production and Characteristics of Raw-Starch-Digesting α-Amylase from a Protease-Negative Aspergillus ficum Mutant

    PubMed Central

    Hayashida, Shinsaku; Teramoto, Yuji

    1986-01-01

    Mutational experiments were carried out to decrease the protease productivity of Aspergillus ficum IFO 4320 by using N-methyl-N′-nitro-N-nitrosoguanidine. A protease-negative mutant, M-33, exhibited higher α-amylaseactivity than the parent strain under submerged culture at 30°C for 24 h. About 70% of the total α-amylase activity in the M-33 culture filtrate was adsorbed onto starch granules. The electrophoretically homogeneous preparation of raw-starch-adsorbable α-amylase (molecular weight, 88,000), acid stable at pH 2, showed intensive raw-starch-digesting activity, dissolving corn starch granules completely. The preparation also exhibited a high synergistic effect with glucoamylase I. A mutant, M-72, with higher protease activity produced a raw cornstarch-unadsorbable α-amylase. The purified enzyme (molecular weight, 54,000), acid unstable, showed no digesting activity on raw corn starch and a lower synergistic effect with glucoamylase I in the hydrolysis of raw corn starch. The fungal α-amylase was therefore divided into two types, a novel type of raw-starch-digesting enzyme and a conventional type of raw-starch-nondigesting enzyme. Images PMID:16347204

  2. Esterification of Starch in Ionic Liquids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We shall discuss the use of various ionic liquids in the preparation of starch esters. Starch was reacted with vinyl acetate in different 1-butyl-3-methylimidazolium (bmim) salts as solvents in an effort to produce starches with different acetylation patterns. Overall degree of substitution (DS) w...

  3. Studies of Amylose Content in Potato Starch

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Potato starch is typically low in amylose (~20-25%), but high amylose starch has superior nutritional qualities. The ratio between amylose and amylopectin is the most important property influencing the physical properties of starch. There is a strong case to be made for the development of food crops...

  4. Surface binding sites in amylase have distinct roles in recognition of starch structure motifs and degradation.

    PubMed

    Cockburn, Darrell; Nielsen, Morten M; Christiansen, Camilla; Andersen, Joakim M; Rannes, Julie B; Blennow, Andreas; Svensson, Birte

    2015-04-01

    Carbohydrate converting enzymes often possess extra substrate binding regions that enhance their activity. These can be found either on separate domains termed carbohydrate binding modules or as so-called surface binding sites (SBSs) situated on the catalytic domain. SBSs are common in starch degrading enzymes and critically important for their function. The affinity towards a variety of starch granules as well as soluble poly- and oligosaccharides of barley α-amylase 1 (AMY1) wild-type and mutants of two SBSs (SBS1 and SBS2) was investigated using Langmuir binding analysis, confocal laser scanning microscopy, affinity gel electrophoresis and surface plasmon resonance to unravel functional roles of the SBSs. SBS1 was critical for binding to different starch types as Kd increased by 7-62-fold or was not measurable upon mutation. By contrast SBS2 was particularly important for binding to soluble polysaccharides and oligosaccharides with α-1,6 linkages, suggesting that branch points are key structural elements in recognition by SBS2. Mutation at both SBS1 and SBS2 eliminated binding to all starch granule types tested. Taken together, the findings indicate that the two SBSs act in concert to localize AMY1 to the starch granule surface and that SBS2 works synergistically with the active site in the degradation of amylopectin.

  5. Properties of retrograded and acetylated starch produced via starch extrusion or starch hydrolysis with pullulanase.

    PubMed

    Kapelko, M; Zięba, T; Gryszkin, A; Styczyńska, M; Wilczak, A

    2013-09-12

    The aim of the present study was to determine the impact of serial modifications of starch, including firstly starch extrusion or hydrolysis with pullulanase, followed by retrogradation (through freezing and defrosting of pastes) and acetylation (under industrial conditions), on its susceptibility to amylolysis. The method of production had a significant effect on properties of the resultant preparations, whilst the direction and extent of changes depended on the type of modification applied. In the produced starch esters, the degree of substitution, expressed by the per cent of acetylation, ranged from 3.1 to 4.4 g/100 g. The acetylation had a significant impact on contents of elements determined with the atomic emission spectrometry, as it contributed to an increased Na content and decreased contents of Ca and K. The DSC thermal characteristics enabled concluding that the modifications caused an increase in temperatures and a decrease in heat of transition (or its lack). The acetylation of retrograded starch preparations increased their solubility in water and water absorbability. The modifications were found to exert various effects on the rheological properties of pastes determined based on the Brabender's pasting characteristics and flow curves determined with the use of an oscillatory-rotating viscosimeter. All starch acetates produced were characterized by ca. 40% resistance to amylolysis.

  6. Redesign of the Active Site of Sucrose Phosphorylase through a Clash-Induced Cascade of Loop Shifts.

    PubMed

    Kraus, Michael; Grimm, Clemens; Seibel, Jürgen

    2016-01-01

    Sucrose phosphorylases have been applied in the enzymatic production of glycosylated compounds for decades. However, several desirable acceptors, such as flavonoids or stilbenoids, that exhibit diverse antimicrobial, anticarcinogenic or antioxidant properties, remain poor substrates. The Q345F exchange in sucrose phosphorylase from Bifidobacterium adolescentis allows efficient glucosylation of resveratrol, (+)-catechin and (-)-epicatechin in yields of up to 97 % whereas the wild-type enzyme favours sucrose hydrolysis. Three previously undescribed products are made available. The crystal structure of the variant reveals a widened access channel with a hydrophobic aromatic surface that is likely to contribute to the improved activity towards aromatic acceptors. The generation of this channel can be explained in terms of a cascade of structural changes arising from the Q345F exchange. The observed mechanisms are likely to be relevant for the design of other tailor-made enzymes.

  7. Synthesis of α(1→4)-linked non-natural mannoglucans by α-glucan phosphorylase-catalyzed enzymatic copolymerization.

    PubMed

    Baba, Ryotaro; Yamamoto, Kazuya; Kadokawa, Jun-Ichi

    2016-10-20

    α-Glucan phosphorylase catalyzes enzymatic polymerization of α-d-glucose 1-phosphate (Glc-1-P) as a monomer from a maltooligosaccharide primer to produce α(1→4)-glucan, i.e., amylose, with liberating inorganic phosphate (Pi). Because of quite weak specificity for the recognition of substrates by thermostable α-glucan phosphorylase (from Aquifex aeolicus VF5), in this study, we investigated the enzymatic copolymerization of Glc-1-P with its analogue monomer, α-d-mannose 1-phosphate (Man-1-P) under the conditions for removal of Pi as the precipitate with ammonium and magnesium in ammonia buffer containing Mg(2+) ion to produce α(1→4)-linked non-natural mannoglucans composed of Glc/Man units. The reaction was conducted in different feed ratios using the maltotriose primer at 40°C for 7days. The MALDI-TOF mass and (1)H NMR spectra of the products fully supported the mannoglucan structures.

  8. Starch synthesis in Arabidopsis is achieved by spatial cotranscription of core starch metabolism genes.

    PubMed

    Tsai, Huang-Lung; Lue, Wei-Ling; Lu, Kuan-Jen; Hsieh, Ming-Hsiun; Wang, Shue-Mei; Chen, Jychian

    2009-11-01

    Starch synthesis and degradation require the participation of many enzymes, occur in both photosynthetic and nonphotosynthetic tissues, and are subject to environmental and developmental regulation. We examine the distribution of starch in vegetative tissues of Arabidopsis (Arabidopsis thaliana) and the expression of genes encoding core enzymes for starch synthesis. Starch is accumulated in plastids of epidermal, mesophyll, vascular, and root cap cells but not in root proper cells. We also identify cells that can synthesize starch heterotrophically in albino mutants. Starch synthesis in leaves is regulated by developmental stage and light. Expression of gene promoter-beta-glucuronidase fusion constructs in transgenic seedlings shows that starch synthesis genes are transcriptionally active in cells with starch synthesis and are inactive in root proper cells except the plastidial phosphoglucose isomerase. In addition, ADG2 (for ADPG PYROPHOSPHORYLASE2) is not required for starch synthesis in root cap cells. Expression profile analysis reveals that starch metabolism genes can be clustered into two sets based on their tissue-specific expression patterns. Starch distribution and expression pattern of core starch synthesis genes are common in Arabidopsis and rice (Oryza sativa), suggesting that the regulatory mechanism for starch metabolism genes may be conserved evolutionarily. We conclude that starch synthesis in Arabidopsis is achieved by spatial coexpression of core starch metabolism genes regulated by their promoter activities and is fine-tuned by cell-specific endogenous and environmental controls.

  9. Pullulanase and Starch Synthase III Are Associated with Formation of Vitreous Endosperm in Quality Protein Maize

    PubMed Central

    Wu, Hao; Clay, Kasi; Thompson, Stephanie S.; Hennen-Bierwagen, Tracie A.; Andrews, Bethany J.; Zechmann, Bernd; Gibbon, Bryan C.

    2015-01-01

    The opaque-2 (o2) mutation of maize increases lysine content, but the low seed density and soft texture of this type of mutant are undesirable. Lines with modifiers of the soft kernel phenotype (mo2) called “Quality Protein Maize” (QPM) have high lysine and kernel phenotypes similar to normal maize. Prior research indicated that the formation of vitreous endosperm in QPM might involve changes in starch granule structure. In this study, we focused on analysis of two starch biosynthetic enzymes that may influence kernel vitreousness. Analysis of recombinant inbred lines derived from a cross of W64Ao2 and K0326Y revealed that pullulanase activity had significant positive correlation with kernel vitreousness. We also found that decreased Starch Synthase III abundance may decrease the pullulanase activity and average glucan chain length given the same Zpu1 genotype. Therefore, Starch Synthase III could indirectly influence the kernel vitreousness by affecting pullulanase activity and coordinating with pullulanase to alter the glucan chain length distribution of amylopectin, resulting in different starch structural properties. The glucan chain length distribution had strong positive correlation with the polydispersity index of glucan chains, which was positively associated with the kernel vitreousness based on nonlinear regression analysis. Therefore, we propose that pullulanase and Starch Synthase III are two important factors responsible for the formation of the vitreous phenotype of QPM endosperms. PMID:26115014

  10. Functional characterization of sucrose phosphorylase and scrR, a regulator of sucrose metabolism in Lactobacillus reuteri.

    PubMed

    Teixeira, Januana S; Abdi, Reihaneh; Su, Marcia Shu-Wei; Schwab, Clarissa; Gänzle, Michael G

    2013-12-01

    Lactobacillus reuteri harbours alternative enzymes for sucrose metabolism, sucrose phosphorylase, fructansucrases, and glucansucrases. Sucrose phosphorylase and fructansucrases additionally contribute to raffinose metabolism. Glucansucrases and fructansucrases produce exopolysaccharides as alternative to sucrose hydrolysis. L. reuteri LTH5448 expresses a levansucrase (ftfA) and sucrose phosphorylase (scrP), both are inducible by sucrose. This study determined the contribution of scrP to sucrose and raffinose metabolism in L. reuteri LTH5448, and elucidated the role of scrR in regulation sucrose metabolism. Disruption of scrP and scrR was achieved by double crossover mutagenesis. L. reuteri LTH5448, LTH5448ΔscrP and LTH5448ΔscrR were characterized with respect to growth and metabolite formation with glucose, sucrose, or raffinose as sole carbon source. Inactivation of scrR led to constitutive transcription of scrP and ftfA, demonstrating that scrR is negative regulator. L. reuteri LTH5448 and the LTH5448ΔscrP or LTH5448ΔscrR mutant strains did not differ with respect to glucose, sucrose or raffinose utilization. However, L. reuteri LTH5448ΔscrP produced more levan, indicating that the lack of sucrose phosphorylase is compensated by an increased metabolic flux through levansucrase. In conclusion, the presence of alternate pathways for sucrose and raffinose metabolism and their regulation indicate that these substrates, which are abundant in plants, are preferred carbohydrate sources for L. reuteri. PMID:24010626

  11. Characterization of the primary starch utilization operon in the obligate anaerobe Bacteroides fragilis: Regulation by carbon source and oxygen.

    PubMed

    Spence, Cheryl; Wells, W Greg; Smith, C Jeffrey

    2006-07-01

    The opportunistic pathogen Bacteroides fragilis is a commensal organism in the large intestine, where it utilizes both dietary and host-derived polysaccharides as a source of carbon and energy. In this study, a four-gene operon required for starch utilization was identified. The operon also was found to be oxygen responsive and thus was designated osu for oxygen-induced starch utilization. The first three genes in the operon were predicted to encode outer membrane proteins involved in starch binding, and a fourth gene, osuD, encoded an amylase involved in starch hydrolysis. Insertional mutation of the osuA gene (Omega osuA) resulted in the inability to utilize starch or glycogen and an insertional mutation into the osuD gene (Omega osuD) was severely impaired for growth on starch media. Transcriptional studies indicated that maltose, maltooligosaccharides, and starch were inducers of osu expression and that maltose was the strongest inducer. A transcriptional activator of osuABCD, OsuR, was identified and found to mediate maltose induction. The Omega osuA and Omega osuD mutants were able to grow on maltose but not starch, whereas a mutation in osuR abolished growth on both substrates, indicating that additional genes under the control of OsuR are needed for maltose utilization. The osuABCD operon also was induced by exposure to oxygen and was shown to be part of the oxidative stress response important for aerotolerance of B. fragilis. Transcriptional analyses showed that osuA was induced 20-fold by oxygen, but OsuR was not required for this activation. Analysis of osu mutants suggested that expression of the operon was important for survival during oxygen exposure but not to hydrogen peroxide stress.

  12. Starch characteristics influencing resistant starch content of cooked buckwheat groats

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Enzyme resistant starch (RS), owing to its health benefits such as colon cancer inhibition, reduced glycemic response, reduced cholesterol level, prevention of gall stone formation and obesity, has received an increasing attention from consumers and food manufacturers, whereas intrinsic and extrinsi...

  13. The binding of D-gluconohydroximo-1,5-lactone to glycogen phosphorylase. Kinetic, ultracentrifugation and crystallographic studies.

    PubMed Central

    Papageorgiou, A C; Oikonomakos, N G; Leonidas, D D; Bernet, B; Beer, D; Vasella, A

    1991-01-01

    Combined kinetic, ultracentrifugation and X-ray-crystallographic studies have characterized the effect of the beta-glucosidase inhibitor gluconohydroximo-1,5-lactone on the catalytic and structural properties of glycogen phosphorylase. In the direction of glycogen synthesis, gluconohydroximo-1,5-lactone was found to competitively inhibit both the b (Ki 0.92 mM) and the alpha form of the enzyme (Ki 0.76 mM) with respect to glucose 1-phosphate in synergism with caffeine. In the direction of glycogen breakdown, gluconohydroximo-1,5-lactone was found to inhibit phosphorylase b in a non-competitive mode with respect to phosphate, and no synergism with caffeine could be demonstrated. Ultracentrifugation and crystallization experiments demonstrated that gluconohydroximo-1,5-lactone was able to induce dissociation of tetrameric phosphorylase alpha and stabilization of the dimeric T-state conformation. A crystallographic binding study with 100 mM-gluconohydroximo-1,5-lactone at 0.24 nm (2.4 A) resolution showed a major peak at the catalytic site, and no significant conformational changes were observed. Analysis of the electron-density map indicated that the ligand adopts a chair conformation. The results are discussed with reference to the ability of the catalytic site of the enzyme to distinguish between two or more conformations of the glucopyranose ring. PMID:1900987

  14. Discovery of Two β-1,2-Mannoside Phosphorylases Showing Different Chain-Length Specificities from Thermoanaerobacter sp. X-514

    PubMed Central

    Suzuki, Erika; Nishimoto, Mamoru; Kitaoka, Motomitsu; Ohtsubo, Ken'ichi; Nakai, Hiroyuki

    2014-01-01

    We characterized Teth514_1788 and Teth514_1789, belonging to glycoside hydrolase family 130, from Thermoanaerobacter sp. X-514. These two enzymes catalyzed the synthesis of 1,2-β-oligomannan using β-1,2-mannobiose and d-mannose as the optimal acceptors, respectively, in the presence of the donor α-d-mannose 1-phosphate. Kinetic analysis of the phosphorolytic reaction toward 1,2-β-oligomannan revealed that these enzymes followed a typical sequential Bi Bi mechanism. The kinetic parameters of the phosphorolysis of 1,2-β-oligomannan indicate that Teth514_1788 and Teth514_1789 prefer 1,2-β-oligomannans containing a DP ≥3 and β-1,2-Man2, respectively. These results indicate that the two enzymes are novel inverting phosphorylases that exhibit distinct chain-length specificities toward 1,2-β-oligomannan. Here, we propose 1,2-β-oligomannan:phosphate α-d-mannosyltransferase as the systematic name and 1,2-β-oligomannan phosphorylase as the short name for Teth514_1788 and β-1,2-mannobiose:phosphate α-d-mannosyltransferase as the systematic name and β-1,2-mannobiose phosphorylase as the short name for Teth514_1789. PMID:25500577

  15. Crystallization and preliminary X-ray study of Vibrio cholerae uridine phosphorylase in complex with 6-methyluracil

    PubMed Central

    Prokofev, Igor I.; Lashkov, Alexander A.; Gabdulkhakov, Azat G.; Dontsova, Mariya V.; Seregina, Tatyana A.; Mironov, Alexander S.; Betzel, Christian; Mikhailov, Al’bert M.

    2014-01-01

    Uridine phosphorylase catalyzes the phosphorolysis of ribonucleosides, with the nitrogenous base and ribose 1-phosphate as products. Additionally, it catalyzes the reverse reaction of the synthesis of ribonucleosides from ribose 1-phosphate and a nitrogenous base. However, the enzyme does not catalyze the synthesis of nucleosides when the substrate is a nitrogenous base substituted at the 6-­position, such as 6-methyluracil (6-MU). In order to explain this fact, it is essential to investigate the three-dimensional structure of the complex of 6-MU with uridine phosphorylase. 6-MU is a pharmaceutical agent that improves tissue nutrition and enhances cell regeneration by normalization of nucleotide exchange in humans. 6-MU is used for the treatment of diseases of the gastrointestinal tract, including infectious diseases. Here, procedures to obtain the uridine phosphorylase from the pathogenic bacterium Vibrio cholerae (VchUPh), purification of this enzyme, crystallization of the complex of VchUPh with 6-MU, and X-ray data collection and preliminary X-ray analysis of the VchUPh–6-MU complex at atomic resolution are reported. PMID:24419619

  16. Measurement of the turnover of glycogen phosphorylase by GC/MS using stable isotope derivatives of pyridoxine (vitamin B6).

    PubMed Central

    Beynon, R J; Leyland, D M; Evershed, R P; Edwards, R H; Coburn, S P

    1996-01-01

    The majority of vitamin B6 in the body is in skeletal muscle, bound as the cofactor pyridoxal 5'-phosphate to one abundant protein, glycogen phosphorylase. Previous work has established that radiolabelled vitamin B6 can be used as a turnover label for glycogen phosphorylase. In this study, a stable isotope derivative of pyridoxine ¿dideuterated pyridoxine; 3-hydroxy-4-(hydroxymethyl) -5-[hydroxymethyl-2H2]-2-methylpyridine¿ ([2H2]PN) has been used as a metabolic tracer to study the kinetics of labelling of the body pools of vitamin B6 in mice. A non-invasive method was developed in which the isotope abundance of the urinary excretory product of vitamin B6 metabolism, 4-pyridoxic acid, was analysed by GC/MS. The change in isotope abundance of urinary 4-pyridoxic acid following administration of [2H2]PN reflects the kinetics of labelling of the body pools of vitamin B6, and yields, non-invasively, the rate of degradation of glycogen phosphorylase. PMID:8713093

  17. Self-association of the alpha subunit of phosphorylase kinase as determined by two-hybrid screening.

    PubMed

    Ayers, N A; Wilkinson, D A; Fitzgerald, T J; Carlson, G M

    1999-12-10

    The structural organization of the (alphabetagammadelta)(4) phosphorylase kinase complex has been studied using the yeast two-hybrid screen for the purpose of elucidating regions of alpha subunit interactions. By screening a rabbit skeletal muscle cDNA library with residues 1-1059 of the alpha subunit of phosphorylase kinase, we have isolated 16 interacting, independent, yet overlapping transcripts of the alpha subunit containing its C-terminal region. Domain mapping of binary interactions between alpha constructs revealed two regions involved in the self-association of the alpha subunit: residues 833-854, a previously unrecognized leucine zipper, and an unspecified region within residues 1015-1237. The cognate binding partner for the latter domain has been inferred to lie within the stretch from residues 864-1059. Indirect evidence from the literature suggests that the interacting domains contained within the latter two, overlapping regions may be further narrowed to the stretches from 1057 to 1237 and from 864 to 971. Cross-linking of the nonactivated holoenzyme with N-(gamma-maleimidobutyroxy)sulfosuccin-imide ester produced intramolecularly cross-linked alpha-alpha dimers, consistent with portions of two alpha subunits in the holoenyzme being in sufficient proximity to associate. This is the first report to identify potential areas of contact between the alpha subunits of phosphorylase kinase. Additionally, issues regarding the general utility of two-hybrid screening as a method for studying homodimeric interactions are discussed. PMID:10585434

  18. A purine nucleoside phosphorylase in Solanum tuberosum L. (potato) with specificity for cytokinins contributes to the duration of tuber endodormancy.

    PubMed

    Bromley, Jennifer R; Warnes, Barbara J; Newell, Christine A; Thomson, Jamie C P; James, Celia M; Turnbull, Colin G N; Hanke, David E

    2014-03-01

    StCKP1 (Solanum tuberosum cytokinin riboside phosphorylase) catalyses the interconversion of the N9-riboside form of the plant hormone CK (cytokinin), a subset of purines, with its most active free base form. StCKP1 prefers CK to unsubstituted aminopurines. The protein was discovered as a CK-binding activity in extracts of tuberizing potato stolon tips, from which it was isolated by affinity chromatography. The N-terminal amino acid sequence matched the translation product of a set of ESTs, enabling a complete mRNA sequence to be obtained by RACE-PCR. The predicted polypeptide includes a cleavable signal peptide and motifs for purine nucleoside phosphorylase activity. The expressed protein was assayed for purine nucleoside phosphorylase activity against CKs and adenine/adenosine. Isopentenyladenine, trans-zeatin, dihydrozeatin and adenine were converted into ribosides in the presence of ribose 1-phosphate. In the opposite direction, isopentenyladenosine, trans-zeatin riboside, dihydrozeatin riboside and adenosine were converted into their free bases in the presence of Pi. StCKP1 had no detectable ribohydrolase activity. Evidence is presented that StCKP1 is active in tubers as a negative regulator of CKs, prolonging endodormancy by a chill-reversible mechanism.

  19. Starch--value addition by modification.

    PubMed

    Tharanathan, Rudrapatnam N

    2005-01-01

    Starch is one of the most important but flexible food ingredients possessing value added attributes for innumerable industrial applications. Its various chemically modified derivatives offer a great scope of high technological value in both food and non-food industries. Modified starches are designed to overcome one or more of the shortcomings, such as loss of viscosity and thickening power upon cooking and storage, particularly at low pH, retrogradation characteristics, syneresis, etc., of native starches. Oxidation, esterification, hydroxyalkylation, dextrinization, and cross-linking are some of the modifications commonly employed to prepare starch derivatives. In a way, starch modification provides desirable functional attributes as well as offering economic alternative to other hydrocolloid ingredients, such as gums and mucilages, which are unreliable in quality and availability. Resistant starch, a highly retrograded starch fractionformed upon food processing, is another useful starch derivative. It exhibits the beneficial physiological effects of therapeutic and nutritional values akin to dietary fiber. There awaits considerable opportunity for future developments, especially for tailor-made starch derivatives with multiple modifications and with the desired functional and nutritional properties, although the problem of obtaining legislative approval for the use of novel starch derivatives in processed food formulations is still under debate. Nevertheless, it can be predicted that new ventures in starch modifications and their diverse applications will continue to be of great interest in applied research.

  20. Resistant starch: promise for improving human health.

    PubMed

    Birt, Diane F; Boylston, Terri; Hendrich, Suzanne; Jane, Jay-Lin; Hollis, James; Li, Li; McClelland, John; Moore, Samuel; Phillips, Gregory J; Rowling, Matthew; Schalinske, Kevin; Scott, M Paul; Whitley, Elizabeth M

    2013-11-01

    Ongoing research to develop digestion-resistant starch for human health promotion integrates the disciplines of starch chemistry, agronomy, analytical chemistry, food science, nutrition, pathology, and microbiology. The objectives of this research include identifying components of starch structure that confer digestion resistance, developing novel plants and starches, and modifying foods to incorporate these starches. Furthermore, recent and ongoing studies address the impact of digestion-resistant starches on the prevention and control of chronic human diseases, including diabetes, colon cancer, and obesity. This review provides a transdisciplinary overview of this field, including a description of types of resistant starches; factors in plants that affect digestion resistance; methods for starch analysis; challenges in developing food products with resistant starches; mammalian intestinal and gut bacterial metabolism; potential effects on gut microbiota; and impacts and mechanisms for the prevention and control of colon cancer, diabetes, and obesity. Although this has been an active area of research and considerable progress has been made, many questions regarding how to best use digestion-resistant starches in human diets for disease prevention must be answered before the full potential of resistant starches can be realized.

  1. Mechanical properties and solubility in water of corn starch-collagen composite films: Effect of starch type and concentrations.

    PubMed

    Wang, Kun; Wang, Wenhang; Ye, Ran; Liu, Anjun; Xiao, Jingdong; Liu, Yaowei; Zhao, Yana

    2017-02-01

    This study investigated the possibility of enhancing the properties of collagen with three different maize starches: waxy maize starch, normal starch, and high amylose starch. Scanning electron microscopy images revealed that starch-collagen films had a rougher surface compared to pure collagen films which became smoother upon heating. Amylose starch and normal starch increased the tensile strength of unheated collagen films in both dry and wet states, while all starches increased tensile strength of collagen film by heating. Depending upon the amylose content and starch concentrations, film solubility in water decreased with the addition of starch. DSC thermograms demonstrated that addition of all starches improved the thermal stability of the collagen film. Moreover, X-ray diffraction results indicated that except for high amylose starch, the crystallinity of both starch and collagen was significantly decreased when subject to heating. FTIR spectra indicated that intermolecular interactions between starch and collagen were enhanced upon heating. PMID:27596411

  2. Preparation, characterization and utilization of starch nanoparticles.

    PubMed

    Kim, Hee-Young; Park, Sung Soo; Lim, Seung-Taik

    2015-02-01

    Starch is one of the most abundant biopolymers in nature and is typically isolated from plants in the form of micro-scale granules. Recent studies reported that nano-scale starch particles could be readily prepared from starch granules, which have unique physical properties. Because starch is environmentally friendly, starch nanoparticles are suggested as one of the promising biomaterials for novel utilization in foods, cosmetics, medicines as well as various composites. An overview of the most up-to-date information regarding the starch nanoparticles including the preparation processes and physicochemical characterization will be presented in this review. Additionally, the prospects and outlooks for the industrial utilization of starch nanoparticles will be discussed.

  3. Properties of baked foams from citric acid modified cassava starch and native cassava starch blends.

    PubMed

    Pornsuksomboon, Kanlaya; Holló, Berta Barta; Szécsényi, Katalin Mészáros; Kaewtatip, Kaewta

    2016-01-20

    Starch foams from native cassava starch (NS) and citric acid modified cassava starch (CNS) were prepared using baking processes with blend ratios of 80/20, 60/40, 50/50, 40/60 and 20/80. The density, thickness, morphology, thermal stability and water absorption of the NS, CNS and blended starch foams were determined. The ratio of the two starch components had a significant influence on the density and thickness of the blended starch foams. All blended starch foams showed good water resistance. Moreover, the morphology of the blended starch foam with the NS/CNS ratio of 50/50 showed a more ordered distribution of cell sizes with thicker cell walls than for the NS and CNS foams. The thermal stability of the blended starch foams was somewhat lower than the stability of the NS foam but not to the extent that it affected any potential practical applications.

  4. Starch composites with aconitic acid.

    PubMed

    Gilfillan, William Neil; Doherty, William O S

    2016-05-01

    The aim of this project is to examine the effectiveness of using aconitic acid (AcA), a tricarboxylic acid which contains a carbon/carbon double bond (CC), to enhance the properties of starch-based films. Starch/glycerol cast films were prepared with 0, 2, 5, 10 and 15wt% AcA (starch wt% basis) and the properties analysed. It was shown that AcA acted as both a cross-linking agent and also a strong plasticising agent. The 5wt% AcA derived starch films were the most effectively cross-linked having the lowest solubility (28wt%) and decreased swelling coefficient (35vol.%) by approximately 3 times and 2.4 times respectively compared to the control film submerged in water (23°C). There was also a significant increase in the film elongation at break by approximately 35 times (compared to the control) with the addition of 15wt% AcA, emphasising the plasticising effect of AcA. However, generally there was a reduced tensile strength, softening of the film, and reduced thermal stability with increased amounts of AcA. PMID:26876996

  5. Starch Phosphorylation in Potato Tubers Proceeds Concurrently with de Novo Biosynthesis of Starch.

    PubMed Central

    Nielsen, T. H.; Wischmann, B.; Enevoldsen, K.; Moller, B. L.

    1994-01-01

    The in vivo phosphorylation of starch was studied in Solanum tuberosum cv Dianella and Posmo. Small starch granules contain 25% more ester-bound phosphate per glucose residue than large starch granules. The degree of phosphorylation was found to be almost constant during tuber development. Isolated tuber discs synthesize starch from externally supplied glucose at a significant rate. Tuber discs supplied with glucose and [32P]orthophosphate incorporate radiolabeled phosphorus into the starch. The level of 32P incorporation is proportional to the amount of starch synthesized. The incorporation of 32P from orthophosphate is correlated to de novo synthesis of starch, since the incorporation of 32P is diminished upon inhibition of starch synthesis by fluoride. Based on the amount of [14C]glucose phosphate isolated after hydrolysis of purified starch from tuber discs incubated in the presence of [U-14C]glucose, approximately 0.5% of the glucose residues of the de novo-synthesized starch are phosphorylated. This value is in general agreement with the observed levels of phosphorus in starch accumulated during tuber development. Thus, the enzyme system responsible for starch phosphorylation is fully active in the isolated tuber discs, and the starch phosphorylation proceeds as an integrated part of de novo starch synthesis. PMID:12232190

  6. Activities of starch hydrolytic enzymes and sucrose-phosphate synthase in the stems of rice subjected to water stress during grain filling.

    PubMed

    Yang, J; Zhang, J; Wang, Z; Zhu, Q

    2001-11-01

    To understand the effect of water stress on the remobilization of prestored carbon reserves, the changes in the activities of starch hydrolytic enzymes and sucrose-phosphate synthase (SPS) in the stems of rice (Oryza sativa L.) during grain filling were investigated. Two rice cultivars, showing high lodging-resistance and slow remobilization, were grown in the field and subjected to well-watered (WW, psi(soil)=0) and water-stressed (WS, psi(soil)=-0.05 MPa) treatments 9 d after anthesis (DAA) till maturity. Leaf water potentials of both cultivars markedly decreased during the day as a result of WS treatment, but completely recovered by early morning. WS treatment accelerated the reduction of starch in the stems, promoted the reallocation of prefixed (14)C from the stems to grains, shortened the grain filling period, and increased the grain filling rate. More soluble sugars including sucrose were accumulated in the stems under WS than under WW treatments. Both alpha- and beta-amylase activities were enhanced by the WS, with the former enhanced more than the latter, and were significantly correlated with the concentrations of soluble sugars in the stems. The other two possible starch-breaking enzymes, alpha-glucosidase and starch phosphorylase, showed no significant differences in the activities between the WW and WS treatments. Water stress also increased the SPS activity that is responsible for sucrose production. Both V(limit) and V(max), the activities of the enzyme at limiting and saturating substrate concentrations, were enhanced and the activation state (V(limit)/V(max)) was also increased as a result of the more significant enhancement of V(limit). The enhanced SPS activity was closely correlated with an increase of sucrose accumulation in the stems. The results suggest that the fast hydrolysis of starch and increased carbon remobilization were attributed to the enhanced alpha-amylase activity and the high activation state of SPS when the rice was subjected

  7. Do mutator mutations fuel tumorigenesis?

    PubMed

    Fox, Edward J; Prindle, Marc J; Loeb, Lawrence A

    2013-12-01

    The mutator phenotype hypothesis proposes that the mutation rate of normal cells is insufficient to account for the large number of mutations found in human cancers. Consequently, human tumors exhibit an elevated mutation rate that increases the likelihood of a tumor acquiring advantageous mutations. The hypothesis predicts that tumors are composed of cells harboring hundreds of thousands of mutations, as opposed to a small number of specific driver mutations, and that malignant cells within a tumor therefore constitute a highly heterogeneous population. As a result, drugs targeting specific mutated driver genes or even pathways of mutated driver genes will have only limited anticancer potential. In addition, because the tumor is composed of such a diverse cell population, tumor cells harboring drug-resistant mutations will exist prior to the administration of any chemotherapeutic agent. We present recent evidence in support of the mutator phenotype hypothesis, major arguments against this concept, and discuss the clinical consequences of tumor evolution fueled by an elevated mutation rate. We also consider the therapeutic possibility of altering the rate of mutation accumulation. Most significantly, we contend that there is a need to fundamentally reconsider current approaches to personalized cancer therapy. We propose that targeting cellular pathways that alter the rate of mutation accumulation in tumors will ultimately prove more effective than attempting to identify and target mutant driver genes or driver pathways.

  8. Physicochemical and functional properties of ozone-oxidized starch.

    PubMed

    Chan, Hui T; Bhat, Rajeev; Karim, Alias A

    2009-07-01

    The effects of oxidation by ozone gas on some physicochemical and functional properties of starch (corn, sago, and tapioca) were investigated. Starch in dry powder form was exposed to ozone for 10 min at different ozone generation times (OGTs). Carboxyl and carbonyl contents increased markedly in all starches with increasing OGTs. Oxidation significantly decreased the swelling power of oxidized sago and tapioca starches but increased that of oxidized corn starch. The solubility of tapioca starch decreased and sago starch increased after oxidation. However, there was an insignificant changed in the solubility of oxidized corn starch. Intrinsic viscosity [eta] of all oxidized starches decreased significantly, except for tapioca starch oxidized at 5 min OGT. Pasting properties of the oxidized starches followed different trends as OGTs increased. These results show that under similar conditions of ozone treatment, the extent of starch oxidation varies among different types of starch. PMID:19489606

  9. Molecular evolution accompanying functional divergence of duplicated genes along the plant starch biosynthesis pathway

    PubMed Central

    2014-01-01

    Background Starch is the main source of carbon storage in the Archaeplastida. The starch biosynthesis pathway (sbp) emerged from cytosolic glycogen metabolism shortly after plastid endosymbiosis and was redirected to the plastid stroma during the green lineage divergence. The SBP is a complex network of genes, most of which are members of large multigene families. While some gene duplications occurred in the Archaeplastida ancestor, most were generated during the sbp redirection process, and the remaining few paralogs were generated through compartmentalization or tissue specialization during the evolution of the land plants. In the present study, we tested models of duplicated gene evolution in order to understand the evolutionary forces that have led to the development of SBP in angiosperms. We combined phylogenetic analyses and tests on the rates of evolution along branches emerging from major duplication events in six gene families encoding sbp enzymes. Results We found evidence of positive selection along branches following cytosolic or plastidial specialization in two starch phosphorylases and identified numerous residues that exhibited changes in volume, polarity or charge. Starch synthases, branching and debranching enzymes functional specializations were also accompanied by accelerated evolution. However, none of the sites targeted by selection corresponded to known functional domains, catalytic or regulatory. Interestingly, among the 13 duplications tested, 7 exhibited evidence of positive selection in both branches emerging from the duplication, 2 in only one branch, and 4 in none of the branches. Conclusions The majority of duplications were followed by accelerated evolution targeting specific residues along both branches. This pattern was consistent with the optimization of the two sub-functions originally fulfilled by the ancestral gene before duplication. Our results thereby provide strong support to the so-called “Escape from Adaptive Conflict

  10. Structure of a complex of uridine phosphorylase from Yersinia pseudotuberculosis with the modified bacteriostatic antibacterial drug determined by X-ray crystallography and computer analysis

    NASA Astrophysics Data System (ADS)

    Balaev, V. V.; Lashkov, A. A.; Gabdoulkhakov, A. G.; Seregina, T. A.; Dontsova, M. V.; Mikhailov, A. M.

    2015-03-01

    Pseudotuberculosis and bubonic plague are acute infectious diseases caused by the bacteria Yersinia pseudotuberculosis and Yersinia pestis. These diseases are treated, in particular, with trimethoprim and its modified analogues. However, uridine phosphorylases (pyrimidine nucleoside phosphorylases) that are present in bacterial cells neutralize the action of trimethoprim and its modified analogues on the cells. In order to reveal the character of the interaction of the drug with bacterial uridine phosphorylase, the atomic structure of the unligated molecule of uridine-specific pyrimidine nucleoside phosphorylase from Yersinia pseudotuberculosis ( YptUPh) was determined by X-ray diffraction at 1.7 Å resolution with high reliability ( R work = 16.2, R free = 19.4%; r.m.s.d. of bond lengths and bond angles are 0.006 Å and 1.005°, respectively; DPI = 0.107 Å). The atoms of the amino acid residues of the functionally important secondary-structure elements—the loop L9 and the helix H8—of the enzyme YptUPh were located. The three-dimensional structure of the complex of YptUPh with modified trimethoprim—referred to as 53I—was determined by the computer simulation. It was shown that 53I is a pseudosubstrate of uridine phosphorylases, and its pyrimidine-2,4-diamine group is located in the phosphate-binding site of the enzyme YptUPh.

  11. Structure of a complex of uridine phosphorylase from Yersinia pseudotuberculosis with the modified bacteriostatic antibacterial drug determined by X-ray crystallography and computer analysis

    SciTech Connect

    Balaev, V. V.; Lashkov, A. A. Gabdoulkhakov, A. G.; Seregina, T. A.; Dontsova, M. V.; Mikhailov, A. M.

    2015-03-15

    Pseudotuberculosis and bubonic plague are acute infectious diseases caused by the bacteria Yersinia pseudotuberculosis and Yersinia pestis. These diseases are treated, in particular, with trimethoprim and its modified analogues. However, uridine phosphorylases (pyrimidine nucleoside phosphorylases) that are present in bacterial cells neutralize the action of trimethoprim and its modified analogues on the cells. In order to reveal the character of the interaction of the drug with bacterial uridine phosphorylase, the atomic structure of the unligated molecule of uridine-specific pyrimidine nucleoside phosphorylase from Yersinia pseudotuberculosis (YptUPh) was determined by X-ray diffraction at 1.7 Å resolution with high reliability (R{sub work} = 16.2, R{sub free} = 19.4%; r.m.s.d. of bond lengths and bond angles are 0.006 Å and 1.005°, respectively; DPI = 0.107 Å). The atoms of the amino acid residues of the functionally important secondary-structure elements—the loop L9 and the helix H8—of the enzyme YptUPh were located. The three-dimensional structure of the complex of YptUPh with modified trimethoprim—referred to as 53I—was determined by the computer simulation. It was shown that 53I is a pseudosubstrate of uridine phosphorylases, and its pyrimidine-2,4-diamine group is located in the phosphate-binding site of the enzyme YptUPh.

  12. Physicochemical Properties of Starch Isolated from Bracken (Pteridium aquilinim) Rhizome.

    PubMed

    Yu, Xurun; Wang, Jin; Zhang, Jing; Wang, Leilei; Wang, Zhong; Xiong, Fei

    2015-12-01

    Bracken (Pteridium aquilinum) is an important wild plant starch resource worldwide. In this work, starch was separated from bracken rhizome, and the physicochemical properties of this starch were systematically investigated and compared with 2 other common starches, that is, starches from waxy maize and potato. There were significant differences in shape, birefringence patterns, size distribution, and amylose content between bracken and the 2 other starches. X-ray diffraction analysis revealed that bracken starch exhibited a typical C-type crystalline structure. Bracken starch presented, respectively, lower and higher relative degree of crystallinity than waxy maize and potato starches. Ordered structures in particle surface differed among these 3 starches. The swelling power tendency of bracken starch in different temperature intervals was very similar to that of potato starch. The viscosity parameters during gelatinization were the lowest in waxy maize, followed by bracken and potato starches. The contents of 3 nutritional components, that is, rapidly digestible, slowly digestible, and resistant starches in native, gelatinized, and retrograded starch from bracken rhizome presented more similarities with potato starch than waxy maize starch. These finding indicated that physicochemical properties of bracken starch showed more similarities with potato starch than waxy maize starch.

  13. Rheological and textural properties of pulse starch gels

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The properties of starch gels from black beans, chickpeas, lentils and navy beans were investigated. Differences were shown between starch sources, and effect of starch concentration was studied. Navy bean starch had the highest peak and final viscosities in pasting tests, while black bean starch h...

  14. Physicochemical Properties of Starch Isolated from Bracken (Pteridium aquilinim) Rhizome.

    PubMed

    Yu, Xurun; Wang, Jin; Zhang, Jing; Wang, Leilei; Wang, Zhong; Xiong, Fei

    2015-12-01

    Bracken (Pteridium aquilinum) is an important wild plant starch resource worldwide. In this work, starch was separated from bracken rhizome, and the physicochemical properties of this starch were systematically investigated and compared with 2 other common starches, that is, starches from waxy maize and potato. There were significant differences in shape, birefringence patterns, size distribution, and amylose content between bracken and the 2 other starches. X-ray diffraction analysis revealed that bracken starch exhibited a typical C-type crystalline structure. Bracken starch presented, respectively, lower and higher relative degree of crystallinity than waxy maize and potato starches. Ordered structures in particle surface differed among these 3 starches. The swelling power tendency of bracken starch in different temperature intervals was very similar to that of potato starch. The viscosity parameters during gelatinization were the lowest in waxy maize, followed by bracken and potato starches. The contents of 3 nutritional components, that is, rapidly digestible, slowly digestible, and resistant starches in native, gelatinized, and retrograded starch from bracken rhizome presented more similarities with potato starch than waxy maize starch. These finding indicated that physicochemical properties of bracken starch showed more similarities with potato starch than waxy maize starch. PMID:26551243

  15. A novel factor FLOURY ENDOSPERM2 is involved in regulation of rice grain size and starch quality.

    PubMed

    She, Kao-Chih; Kusano, Hiroaki; Koizumi, Kazuyoshi; Yamakawa, Hiromoto; Hakata, Makoto; Imamura, Tomohiro; Fukuda, Masato; Naito, Natsuka; Tsurumaki, Yumi; Yaeshima, Mitsuhiro; Tsuge, Tomohiko; Matsumoto, Ken'ichiro; Kudoh, Mari; Itoh, Eiko; Kikuchi, Shoshi; Kishimoto, Naoki; Yazaki, Junshi; Ando, Tsuyu; Yano, Masahiro; Aoyama, Takashi; Sasaki, Tadamasa; Satoh, Hikaru; Shimada, Hiroaki

    2010-10-01

    Rice (Oryza sativa) endosperm accumulates a massive amount of storage starch and storage proteins during seed development. However, little is known about the regulatory system involved in the production of storage substances. The rice flo2 mutation resulted in reduced grain size and starch quality. Map-based cloning identified FLOURY ENDOSPERM2 (FLO2), a member of a novel gene family conserved in plants, as the gene responsible for the rice flo2 mutation. FLO2 harbors a tetratricopeptide repeat motif, considered to mediate a protein-protein interactions. FLO2 was abundantly expressed in developing seeds coincident with production of storage starch and protein, as well as in leaves, while abundant expression of its homologs was observed only in leaves. The flo2 mutation decreased expression of genes involved in production of storage starch and storage proteins in the endosperm. Differences between cultivars in their responsiveness of FLO2 expression during high-temperature stress indicated that FLO2 may be involved in heat tolerance during seed development. Overexpression of FLO2 enlarged the size of grains significantly. These results suggest that FLO2 plays a pivotal regulatory role in rice grain size and starch quality by affecting storage substance accumulation in the endosperm.

  16. A novel factor FLOURY ENDOSPERM2 is involved in regulation of rice grain size and starch quality.

    PubMed

    She, Kao-Chih; Kusano, Hiroaki; Koizumi, Kazuyoshi; Yamakawa, Hiromoto; Hakata, Makoto; Imamura, Tomohiro; Fukuda, Masato; Naito, Natsuka; Tsurumaki, Yumi; Yaeshima, Mitsuhiro; Tsuge, Tomohiko; Matsumoto, Ken'ichiro; Kudoh, Mari; Itoh, Eiko; Kikuchi, Shoshi; Kishimoto, Naoki; Yazaki, Junshi; Ando, Tsuyu; Yano, Masahiro; Aoyama, Takashi; Sasaki, Tadamasa; Satoh, Hikaru; Shimada, Hiroaki

    2010-10-01

    Rice (Oryza sativa) endosperm accumulates a massive amount of storage starch and storage proteins during seed development. However, little is known about the regulatory system involved in the production of storage substances. The rice flo2 mutation resulted in reduced grain size and starch quality. Map-based cloning identified FLOURY ENDOSPERM2 (FLO2), a member of a novel gene family conserved in plants, as the gene responsible for the rice flo2 mutation. FLO2 harbors a tetratricopeptide repeat motif, considered to mediate a protein-protein interactions. FLO2 was abundantly expressed in developing seeds coincident with production of storage starch and protein, as well as in leaves, while abundant expression of its homologs was observed only in leaves. The flo2 mutation decreased expression of genes involved in production of storage starch and storage proteins in the endosperm. Differences between cultivars in their responsiveness of FLO2 expression during high-temperature stress indicated that FLO2 may be involved in heat tolerance during seed development. Overexpression of FLO2 enlarged the size of grains significantly. These results suggest that FLO2 plays a pivotal regulatory role in rice grain size and starch quality by affecting storage substance accumulation in the endosperm. PMID:20889913

  17. Thermal and rheological properties of breadfruit starch.

    PubMed

    Wang, Xueyu; Chen, Ling; Li, Xiaoxi; Xie, Fengwei; Liu, Hongshen; Yu, Long

    2011-01-01

    The thermal and rheological properties of breadfruit starch were studied using DSC and 2 different rheometers. It was found that the gelatinization temperature of starch with excess moisture content (>70%) was at approximately 75 °C. A new endotherm was detected at about 173 °C when the moisture content was lower than required for full gelatinization of the starch. A detailed examination revealed that this endotherm represented the melting of amylose-lipid complexes. Breadfruit starch paste exhibited shear-thinning fluid characteristics, and good thermal and pH stability. The setback viscosity of the breadfruit starch was lower than that of potato and corn starches. The rheological properties of the breadfruit starch paste was well described by the Herschel-Bulkley model at a shear rate of 0 to 100 s(-1), where R(2) is greater than 0.95, and it behaved like a yield-pseudoplastic fluid. Both the storage modulus and loss modulus of the paste initially increased sharply, then dropped after reaching the gelatinization peak. Breadfruit starch gel showed both flexibility and viscosity. Suspension with 6% starch content exhibited very weak gel rigidity; however, this increased significantly at starch contents above 20%.

  18. Starch hydrolysis by the ruminal microflora.

    PubMed

    Kotarski, S F; Waniska, R D; Thurn, K K

    1992-01-01

    The effects of grain type and processing on ruminal starch digestion are well documented but poorly understood at the biochemical and molecular levels. Waxy grains have starches high in amylopectin and are more readily digested than nonwaxy grains. However, the composition of the endosperm cell matrix and the extent to which the starch granules are embedded within it also affect starch digestion rates. Continued work is needed to determine the influence of specific cell matrix proteins, protein-starch interactions and cell wall carbohydrates on starch availability. The microbial populations that metabolize starch are diverse, differing in their capacities to hydrolyze starch granules and soluble forms of starch. Surveys show that the amylases are under regulatory control in most of these organisms, but few studies have addressed the types of amylolytic enzymes produced, their regulation and the impact of other plant polymers on their synthesis. Research in these areas, coupled with the development and use of isogeneic or near-isogeneic grain cultivars with biochemically defined endosperm characteristics, will enhance our ability to identify mechanisms to manipulate ruminal starch digestion.

  19. Molecular disassembly of starch granules during gelatinization and its effect on starch digestibility: a review.

    PubMed

    Wang, Shujun; Copeland, Les

    2013-11-01

    Starch is the most important glycemic carbohydrate in foods. The relationship between the rate and extent of starch digestion to produce glucose for absorption into the bloodstream and risk factors for diet-related diseases is of considerable nutritional interest. Native starch is attacked slowly by enzymes, but after hydrothermal processing its susceptibility to enzymatic breakdown is greatly increased. Most starch consumed by humans has undergone some form of processing or cooking, which causes native starch granules to gelatinize, followed by retrogradation on cooling. The extent of gelatinization and retrogradation are major determinants of the susceptibility of starch to enzymatic digestion and its functional properties for food processing. The type and extent of changes that occur in starch as a result of gelatinization, pasting and retrogradation are determined by the type of the starch, processing and storage conditions. A mechanistic understanding of the molecular disassembly of starch granules during gelatinization is critical to explaining the effects of processing or cooking on starch digestibility. This review focuses on the molecular disassembly of starch granules during starch gelatinization over a wide range of water levels, and its consequential effect on in vitro starch digestibility and in vivo glycemic index.

  20. Encapsulation altered starch digestion: toward developing starch-based delivery systems.

    PubMed

    Janaswamy, Srinivas

    2014-01-30

    Starch is an abundant biomaterial that forms a vital energy source for humans. Altering its digestion, e.g. increasing the proportions of slowly digestible starch (SDS) and resistant starch (RS), would revolutionize starch utility in addressing a number of health issues related to glucose absorption, glycemic index and colon health. The research reported in this article is based on my hypothesis that water channels present in the B-type starch crystalline matrix, particularly in tuber starches, can embed guest molecules such as nutraceuticals, drugs, flavor compounds and vitamins leading to altered starch digestion. Toward this goal, potato starch has been chosen as the model tuber starch, and ibuprofen, benzocaine, sulfapyridine, curcumin, thymol and ascorbic acid as model guest molecules. X-ray powder diffraction and FT-IR analyses clearly suggest the incorporation of guest molecules in the water channels of potato starch. Furthermore, the in vitro digestion profiles of complexes are intriguing with major variations occurring after 60 min of starch digestion and finally at 120 min. These changes are concomitantly reflected in the SDS and RS amounts, with about 24% decrease in SDS for benzocaine complex and 6% increase in RS for ibuprofen complex, attesting the ability of guest molecule encapsulation in modulating the digestion properties of potato starch. Overall, this research provides an elegant opportunity for the design and development of novel starch-based stable carriers that not only bestow tailored glucose release rates but could also transport health promoting and disease preventing compounds.

  1. Starch and cellulose nanocrystals together into thermoplastic starch bionanocomposites.

    PubMed

    González, Kizkitza; Retegi, Aloña; González, Alba; Eceiza, Arantxa; Gabilondo, Nagore

    2015-03-01

    In the present work, thermoplastic maize starch based bionanocomposites were prepared as transparent films, plasticized with 35% of glycerol and reinforced with both waxy starch (WSNC) and cellulose nanocrystals (CNC), previously extracted by acidic hydrolysis. The influence of the nanofiller content was evaluated at 1 wt.%, 2.5 wt.% and 5 wt.% of WSNC. The effect of adding the two different nanoparticles at 1 wt.% was also investigated. As determined by tensile measurements, mechanical properties were improved at any composition of WSNC. Water vapour permeance values maintained constant, whereas barrier properties to oxygen reduced in a 70%, indicating the effectiveness of hydrogen bonding at the interphase. The use of CNC or CNC and WSNC upgraded mechanical results, but no significant differences in barrier properties were obtained. A homogeneous distribution of the nanofillers was demonstrated by atomic force microscopy, and a shift of the two relaxation peaks to higher temperatures was detected by dynamic mechanical analysis.

  2. Defects in polynucleotide phosphorylase impairs virulence in Escherichia coli O157:H7.

    PubMed

    Hu, Jia; Zhu, Mei-Jun

    2015-01-01

    Polynucleotide phosphorylase (PNPase) is reported to regulate virulence in Salmonella, Yersinia sp. and Campylobacter jejuni, yet its role in Escherichia coli O157:H7 has not been investigated. To gain insights into its roles in E. coli O157:H7 virulence, pnp deletion mutants were generated and the major virulence factors were compared to their parental wild type strains. Deletion of pnp in E. coli O157:H7 dramatically decreased stx2 mRNA expression and Stx2 protein production, and impaired lambdoid prophage activation in E. coli O157:H7. Quantitative PCR further confirmed that the Stx2 phage lytic growth was repressed by pnp deletion. Consistent with reduced Stx2 production and Stx2 phage activation, the transcriptional levels of genes involved in phage lysis and replication were down-regulated. In addition, disruption of pnp in E. coli O157:H7 decreased its adhesion to intestinal epithelial cells as well as cattle colonic explant tissues. On the other hand, PNPase inactivation in E. coli O157:H7 enhanced Tir protein content and the transcription of type three secretion system components, including genes encoding intimin, Tir, and EspB as well as locus of enterocyte and effacement positive regulator, Ler. Collectively, data indicate that PNPase has pleiotropic effects on the virulence of E. coli O157:H7. PMID:26347717

  3. Multiple cellobiohydrolases and cellobiose phosphorylases cooperate in the ruminal bacterium Ruminococcus albus 8 to degrade cellooligosaccharides

    PubMed Central

    Devendran, Saravanan; Abdel-Hamid, Ahmed M.; Evans, Anton F.; Iakiviak, Michael; Kwon, In Hyuk; Mackie, Roderick I.; Cann, Isaac

    2016-01-01

    Digestion of plant cell wall polysaccharides is important in energy capture in the gastrointestinal tract of many herbivorous and omnivorous mammals, including humans and ruminants. The members of the genus Ruminococcus are found in both the ruminant and human gastrointestinal tract, where they show versatility in degrading both hemicellulose and cellulose. The available genome sequence of Ruminococcus albus 8, a common inhabitant of the cow rumen, alludes to a bacterium well-endowed with genes that target degradation of various plant cell wall components. The mechanisms by which R. albus 8 employs to degrade these recalcitrant materials are, however, not clearly understood. In this report, we demonstrate that R. albus 8 elaborates multiple cellobiohydrolases with multi-modular architectures that overall enhance the catalytic activity and versatility of the enzymes. Furthermore, our analyses show that two cellobiose phosphorylases encoded by R. albus 8 can function synergistically with a cognate cellobiohydrolase and endoglucanase to completely release, from a cellulosic substrate, glucose which can then be fermented by the bacterium for production of energy and cellular building blocks. We further use transcriptomic analysis to confirm the over-expression of the biochemically characterized enzymes during growth of the bacterium on cellulosic substrates compared to cellobiose. PMID:27748409

  4. The kinetic mechanism of Human Thymidine Phosphorylase - a molecular target for cancer drug development.

    PubMed

    Deves, Candida; Rostirolla, Diana Carolina; Martinelli, Leonardo Kras Borges; Bizarro, Cristiano Valim; Santos, Diogenes Santiago; Basso, Luiz Augusto

    2014-03-01

    Human Thymidine Phosphorylase (HTP), also known as the platelet-derived endothelial cell growth factor (PD-ECGF) or gliostatin, catalyzes the reversible phosphorolysis of thymidine (dThd) to thymine and 2-deoxy-α-d-ribose-1-phosphate (2dR1P). HTP is a key enzyme in the pyrimidine salvage pathway involved in dThd homeostasis in cells. HTP is a target for anticancer drug development as its enzymatic activity promotes angiogenesis. Here, we describe cloning, expression, and purification to homogeneity of recombinant TYMP-encoded HTP. Peptide fingerprinting and the molecular mass value of the homogenous protein confirmed its identity as HTP assessed by mass spectrometry. Size exclusion chromatography showed that HTP is a dimer in solution. Kinetic studies revealed that HTP displayed substrate inhibition for dThd. Initial velocity and isothermal titration calorimetry (ITC) studies suggest that HTP catalysis follows a rapid-equilibrium random bi-bi kinetic mechanism. ITC measurements also showed that dThd and Pi binding are favorable processes. The pH-rate profiles indicated that maximal enzyme activity was achieved at low pH values. Functional groups with apparent pK values of 5.2 and 9.0 are involved in dThd binding and groups with pK values of 6.1 and 7.8 are involved in phosphate binding. PMID:24407036

  5. Insights into Brain Glycogen Metabolism: THE STRUCTURE OF HUMAN BRAIN GLYCOGEN PHOSPHORYLASE.

    PubMed

    Mathieu, Cécile; de la Sierra-Gallay, Ines Li; Duval, Romain; Xu, Ximing; Cocaign, Angélique; Léger, Thibaut; Woffendin, Gary; Camadro, Jean-Michel; Etchebest, Catherine; Haouz, Ahmed; Dupret, Jean-Marie; Rodrigues-Lima, Fernando

    2016-08-26

    Brain glycogen metabolism plays a critical role in major brain functions such as learning or memory consolidation. However, alteration of glycogen metabolism and glycogen accumulation in the brain contributes to neurodegeneration as observed in Lafora disease. Glycogen phosphorylase (GP), a key enzyme in glycogen metabolism, catalyzes the rate-limiting step of glycogen mobilization. Moreover, the allosteric regulation of the three GP isozymes (muscle, liver, and brain) by metabolites and phosphorylation, in response to hormonal signaling, fine-tunes glycogenolysis to fulfill energetic and metabolic requirements. Whereas the structures of muscle and liver GPs have been known for decades, the structure of brain GP (bGP) has remained elusive despite its critical role in brain glycogen metabolism. Here, we report the crystal structure of human bGP in complex with PEG 400 (2.5 Å) and in complex with its allosteric activator AMP (3.4 Å). These structures demonstrate that bGP has a closer structural relationship with muscle GP, which is also activated by AMP, contrary to liver GP, which is not. Importantly, despite the structural similarities between human bGP and the two other mammalian isozymes, the bGP structures reveal molecular features unique to the brain isozyme that provide a deeper understanding of the differences in the activation properties of these allosteric enzymes by the allosteric effector AMP. Overall, our study further supports that the distinct structural and regulatory properties of GP isozymes contribute to the different functions of muscle, liver, and brain glycogen. PMID:27402852

  6. Rac1 Protein Regulates Glycogen Phosphorylase Activation and Controls Interleukin (IL)-2-dependent T Cell Proliferation*

    PubMed Central

    Arrizabalaga, Onetsine; Lacerda, Hadriano M.; Zubiaga, Ana M.; Zugaza, José L.

    2012-01-01

    Small GTPases of the Rho family have been implicated in important cellular processes such as cell migration and adhesion, protein secretion, and/or gene transcription. In the lymphoid system, these GTPases participate in the signaling cascades that are activated after engagement of antigen receptors. However, little is known about the role that Rho GTPases play in IL-2-mediated responses. Here, we show that IL-2 induces Rac1 activation in Kit 225 T cells. We identified by mass spectrometry the muscle isoform of glycogen phosphorylase (PYGM) as a novel Rac1 effector molecule in IL-2-stimulated cells. The interaction between the active form of Rac1 (Rac1-GTP) and PYGM was established directly through a domain comprising amino acids 191–270 of PYGM that exhibits significant homology with the Rac binding domain of PAK1. The integrity of this region was crucial for PYGM activation. Importantly, IL-2-dependent cellular proliferation was inhibited upon blocking both the activation of Rac1 and the activity of PYGM. These results reveal a new role for Rac1 in cell signaling, showing that this GTPase triggers T cell proliferation upon IL-2 stimulation by associating with PYGM and modulating its enzymatic activity. PMID:22337875

  7. Multiple cellobiohydrolases and cellobiose phosphorylases cooperate in the ruminal bacterium Ruminococcus albus 8 to degrade cellooligosaccharides

    NASA Astrophysics Data System (ADS)

    Devendran, Saravanan; Abdel-Hamid, Ahmed M.; Evans, Anton F.; Iakiviak, Michael; Kwon, In Hyuk; Mackie, Roderick I.; Cann, Isaac

    2016-10-01

    Digestion of plant cell wall polysaccharides is important in energy capture in the gastrointestinal tract of many herbivorous and omnivorous mammals, including humans and ruminants. The members of the genus Ruminococcus are found in both the ruminant and human gastrointestinal tract, where they show versatility in degrading both hemicellulose and cellulose. The available genome sequence of Ruminococcus albus 8, a common inhabitant of the cow rumen, alludes to a bacterium well-endowed with genes that target degradation of various plant cell wall components. The mechanisms by which R. albus 8 employs to degrade these recalcitrant materials are, however, not clearly understood. In this report, we demonstrate that R. albus 8 elaborates multiple cellobiohydrolases with multi-modular architectures that overall enhance the catalytic activity and versatility of the enzymes. Furthermore, our analyses show that two cellobiose phosphorylases encoded by R. albus 8 can function synergistically with a cognate cellobiohydrolase and endoglucanase to completely release, from a cellulosic substrate, glucose which can then be fermented by the bacterium for production of energy and cellular building blocks. We further use transcriptomic analysis to confirm the over-expression of the biochemically characterized enzymes during growth of the bacterium on cellulosic substrates compared to cellobiose.

  8. Cold-temperature induction of Escherichia coli polynucleotide phosphorylase occurs by reversal of its autoregulation.

    PubMed

    Beran, R K; Simons, R W

    2001-01-01

    When Escherichia coli cells are shifted to low temperatures (e.g. 15 degrees C), growth halts while the 'cold shock response' (CSR) genes are induced, after which growth resumes. One CSR gene, pnp, encodes polynucleotide phosphorylase (PNPase), a 3'-exoribonuclease and component of the RNA degradosome. At 37 degrees C, ribonuclease III (RNase III, encoded by rnc) cleaves the pnp untranslated leader, whereupon PNPase represses its own translation by an unknown mechanism. Here, we show that PNPase cold-temperature induction involves several post-transcriptional events, all of which require the intact pnp mRNA leader. The bulk of induction results from reversal of autoregulation at a step subsequent to RNase III cleavage of the pnp leader. We also found that pnp translation occurs throughout cold-temperature adaptation, whereas lacZ(+) translation was delayed. This difference is striking, as both mRNAs are greatly stabilized upon the shift to 15 degrees C. However, unlike the lacZ(+) mRNA, which remains stable during adaptation, pnp mRNA decay accelerates. Together with other evidence, these results suggest that mRNA is generally stabilized upon a shift to cold temperatures, but that a CSR mRNA-specific decay process is initiated during adaptation.

  9. Central nervous system dysfunction and erythrocyte guanosine triphosphate depletion in purine nucleoside phosphorylase deficiency.

    PubMed Central

    Simmonds, H A; Fairbanks, L D; Morris, G S; Morgan, G; Watson, A R; Timms, P; Singh, B

    1987-01-01

    Developmental retardation was a prominent clinical feature in six infants from three kindreds deficient in the enzyme purine nucleoside phosphorylase (PNP) and was present before development of T cell immunodeficiency. Guanosine triphosphate (GTP) depletion was noted in the erythrocytes of all surviving homozygotes and was of equivalent magnitude to that found in the Lesch-Nyhan syndrome (complete hypoxanthine-guanine phosphoribosyltransferase (HGPRT) deficiency). The similarity between the neurological complications in both disorders indicates that the two major clinical consequences of complete PNP deficiency have differing aetiologies: neurological effects resulting from deficiency of the PNP enzyme products, which are the substrates for HGPRT, leading to functional deficiency of this enzyme. immunodeficiency caused by accumulation of the PNP enzyme substrates, one of which, deoxyguanosine, is toxic to T cells. These studies show the need to consider PNP deficiency (suggested by the finding of hypouricaemia) in patients with neurological dysfunction, as well as in T cell immunodeficiency. They suggest an important role for GTP in normal central nervous system function. PMID:2439024

  10. Thymidine phosphorylase exerts complex effects on bone resorption and formation in myeloma.

    PubMed

    Liu, Huan; Liu, Zhiqiang; Du, Juan; He, Jin; Lin, Pei; Amini, Behrang; Starbuck, Michael W; Novane, Nora; Shah, Jatin J; Davis, Richard E; Hou, Jian; Gagel, Robert F; Yang, Jing

    2016-08-24

    Myelomatous bone disease is characterized by the development of lytic bone lesions and a concomitant reduction in bone formation, leading to chronic bone pain and fractures. To understand the underlying mechanism, we investigated the contribution of myeloma-expressed thymidine phosphorylase (TP) to bone lesions. In osteoblast progenitors, TP up-regulated the methylation of RUNX2 and osterix, leading to decreased bone formation. In osteoclast progenitors, TP up-regulated the methylation of IRF8 and thereby enhanced expression of NFATc1 (nuclear factor of activated T cells, cytoplasmic 1 protein), leading to increased bone resorption. TP reversibly catalyzes thymidine into thymine and 2-deoxy-d-ribose (2DDR). Myeloma-secreted 2DDR bound to integrin αVβ3/α5β1 in the progenitors, activated PI3K (phosphoinositide 3-kinase)/Akt signaling, and increased DNMT3A (DNA methyltransferase 3A) expression, resulting in hypermethylation of RUNX2, osterix, and IRF8 This study elucidates an important mechanism for myeloma-induced bone lesions, suggesting that targeting TP may be a viable approach to healing resorbed bone in patients. Because TP overexpression is common in bone-metastatic tumors, our findings could have additional mechanistic implications. PMID:27559096

  11. Isoform-selective regulation of glycogen phosphorylase by energy deprivation and phosphorylation in astrocytes.

    PubMed

    Müller, Margit S; Pedersen, Sofie E; Walls, Anne B; Waagepetersen, Helle S; Bak, Lasse K

    2015-01-01

    Glycogen phosphorylase (GP) is activated to degrade glycogen in response to different stimuli, to support both the astrocyte's own metabolic demand and the metabolic needs of neurons. The regulatory mechanism allowing such a glycogenolytic response to distinct triggers remains incompletely understood. In the present study, we used siRNA-mediated differential knockdown of the two isoforms of GP expressed in astrocytes, muscle isoform (GPMM), and brain isoform (GPBB), to analyze isoform-specific regulatory characteristics in a cellular setting. Subsequently, we tested the response of each isoform to phosphorylation, triggered by incubation with norepinephrine (NE), and to AMP, increased by glucose deprivation in cells in which expression of one GP isoform had been silenced. Successful knockdown was demonstrated on the protein level by Western blot, and on a functional level by determination of glycogen content showing an increase in glycogen levels following knockdown of either GPMM or GPBB. NE triggered glycogenolysis within 15 min in control cells and after GPBB knockdown. However, astrocytes in which expression of GPMM had been silenced showed a delay in response to NE, with glycogen levels significantly reduced only after 60 min. In contrast, allosteric activation of GP by AMP, induced by glucose deprivation, seemed to mainly affect GPBB, as only knockdown of GPBB, but not of GPMM, delayed the glycogenolytic response to glucose deprivation. Our results indicate that the two GP isoforms expressed in astrocytes respond to different physiological triggers, therefore conferring distinct metabolic functions of brain glycogen.

  12. Multiple disulfide bridges modulate conformational stability and flexibility in hyperthermophilic archaeal purine nucleoside phosphorylase.

    PubMed

    Bagarolo, Maria Libera; Porcelli, Marina; Martino, Elisa; Feller, Georges; Cacciapuoti, Giovanna

    2015-10-01

    5'-Deoxy-5'-methylthioadenosine phosphorylase from Sulfolobus solfataricus is a hexameric hyperthermophilic protein containing in each subunit two pairs of disulfide bridges, a CXC motif, and one free cysteine. The contribution of each disulfide bridge to the protein conformational stability and flexibility has been assessed by comparing the thermal unfolding and the limited proteolysis of the wild-type enzyme and its variants obtained by site-directed mutagenesis of the seven cysteine residues. All variants catalyzed efficiently MTA cleavage with specific activity similar to the wild-type enzyme. The elimination of all cysteine residues caused a substantial decrease of ΔHcal (850 kcal/mol) and Tmax (39°C) with respect to the wild-type indicating that all cysteine pairs and especially the CXC motif significantly contribute to the enzyme thermal stability. Disulfide bond Cys200-Cys262 and the CXC motif weakly affected protein flexibility while the elimination of the disulfide bond Cys138-Cys205 lead to an increased protease susceptibility. Experimental evidence from limited proteolysis, differential scanning calorimetry, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions also allowed to propose a stabilizing role for the free Cys164.

  13. Regulation of glycogen metabolism in cultured human muscles by the glycogen phosphorylase inhibitor CP-91149.

    PubMed Central

    Lerín, Carlos; Montell, Eulàlia; Nolasco, Teresa; García-Rocha, Mar; Guinovart, Joan J; Gómez-Foix, Anna M

    2004-01-01

    Pharmacological inhibition of liver GP (glycogen phosphorylase), which is currently being studied as a treatment for Type II (non-insulin-dependent) diabetes, may affect muscle glycogen metabolism. In the present study, we analysed the effects of the GP inhibitor CP-91149 on non-engineered or GP-overexpressing cultured human muscle cells. We found that CP-91149 treatment decreased muscle GP activity by (1) converting the phosphorylated AMP-independent a form into the dephosphorylated AMP-dependent b form and (2) inhibiting GP a activity and AMP-mediated GP b activation. Dephosphorylation of GP was exerted, irrespective of incubation of the cells with glucose, whereas inhibition of its activity was synergic with glucose. As expected, CP-91149 impaired the glycogenolysis induced by glucose deprivation. CP-91149 also promoted the dephosphorylation and activation of GS (glycogen synthase) in non-engineered or GP-overexpressing cultured human muscle cells, but exclusively in glucose-deprived cells. However, this inhibitor did not activate GS in glucose-deprived but glycogen-replete cells overexpressing PTG (protein targeting to glycogen), thus suggesting that glycogen inhibits the CP-91149-mediated activation of GS. Consistently, CP-91149 promoted glycogen resynthesis, but not its overaccumulation. Hence, treatment with CP-91149 impairs muscle glycogen breakdown, but enhances its recovery, which may be useful for the treatment of Type II (insulin-dependent) diabetes. PMID:14651477

  14. Brain isoform glycogen phosphorylase as a novel hepatic progenitor cell marker.

    PubMed

    Huang, Yu-Wen; Chiu, Chien-Chang; Liang, Ja-Der; Chiou, Ling-Ling; Huang, Guan-Tarn; Yu, Ming-Jiun; Lee, Hsuan-Shu

    2015-01-01

    An appropriate liver-specific progenitor cell marker is a stepping stone in liver regenerative medicine. Here, we report brain isoform glycogen phosphorylase (GPBB) as a novel liver progenitor cell marker. GPBB was identified in a protein complex precipitated by a monoclonal antibody Ligab generated from a rat liver progenitor cell line Lig-8. Immunoblotting results show that GPBB was expressed in two liver progenitor cell lines Lig-8 and WB-F344. The levels of GPBB expression decreased in the WB-F344 cells under sodium butyrate (SB)-induced cell differentiation, consistent with roles of GPBB as a liver progenitor cell marker. Short hairpin RNA (shRNA)-mediated GPBB knockdown followed by glucose deprivation test shows that GPBB aids in liver progenitor cell survival under low glucose conditions. Furthermore, shRNA-mediated GPBB knockdown followed by SB-induced cell differentiation shows that reducing GPBB expression delayed liver progenitor cell differentiation. We conclude that GPBB is a novel liver progenitor cell marker, which facilitates liver progenitor cell survival under low glucose conditions and cell differentiation. PMID:25826279

  15. Crystal structure and molecular dynamics studies of human purine nucleoside phosphorylase complexed with 7-deazaguanine.

    PubMed

    Caceres, Rafael Andrade; Timmers, Luis Fernando Saraiva Macedo; Pauli, Ivani; Gava, Lisandra Marques; Ducati, Rodrigo Gay; Basso, Luiz Augusto; Santos, Diógenes Santiago; de Azevedo, Walter Filgueira

    2010-03-01

    In humans, purine nucleoside phosphorylase (HsPNP) is responsible for degradation of deoxyguanosine, and genetic deficiency of this enzyme leads to profound T-cell mediated immunosuppression. HsPNP is a target for inhibitor development aiming at T-cell immune response modulation. Here we report the crystal structure of HsPNP in complex with 7-deazaguanine (HsPNP:7DG) at 2.75 A. Molecular dynamics simulations were employed to assess the structural features of HsPNP in both free form and in complex with 7DG. Our results show that some regions, responsible for entrance and exit of substrate, present a conformational variability, which is dissected by dynamics simulation analysis. Enzymatic assays were also carried out and revealed that 7-deazaguanine presents a lower inhibitory activity against HsPNP (K(i)=200 microM). The present structure may be employed in both structure-based design of PNP inhibitors and in development of specific empirical scoring functions.

  16. Crystal structure and molecular dynamics studies of purine nucleoside phosphorylase from Mycobacterium tuberculosis associated with acyclovir.

    PubMed

    Caceres, Rafael A; Timmers, Luís F S M; Ducati, Rodrigo G; da Silva, Diego O N; Basso, Luiz A; de Azevedo, Walter F; Santos, Diógenes S

    2012-01-01

    Consumption has been a scourge of mankind since ancient times. This illness has charged a high price to human lives. Many efforts have been made to defeat Mycobacterium tuberculosis (Mt). The M. tuberculosis purine nucleoside phosphorylase (MtPNP) is considered an interesting target to pursuit new potential inhibitors, inasmuch it belongs to the purine salvage pathway and its activity might be involved in the mycobacterial latency process. Here we present the MtPNP crystallographic structure associated with acyclovir and phosphate (MtPNP:ACY:PO(4)) at 2.10 Å resolution. Molecular dynamics simulations were carried out in order to dissect MtPNP:ACY:PO(4) structural features, and the influence of the ligand in the binding pocket stability. Our results revealed that the ligand leads to active site lost of stability, in agreement with experimental results, which demonstrate a considerable inhibitory activity against MtPNP (K(i) = 150 nM). Furthermore, we observed that some residues which are important in the proper ligand's anchor into the human homologous enzyme do not present the same importance to MtPNP. Therewithal, these findings contribute to the search of new specific inhibitors for MtPNP, since peculiarities between the mycobacterial and human enzyme binding sites have been identified, making a structural-based drug design feasible.

  17. Substrate specificity and kinetic mechanism of purine nucleoside phosphorylase from Mycobacterium tuberculosis.

    PubMed

    Ducati, Rodrigo G; Santos, Diógenes S; Basso, Luiz A

    2009-06-15

    Purine nucleoside phosphorylase from Mycobacterium tuberculosis (MtPNP) is numbered among targets for persistence of the causative agent of tuberculosis. Here, it is shown that MtPNP is more specific to natural 6-oxopurine nucleosides and synthetic compounds, and does not catalyze the phosphorolysis of adenosine. Initial velocity, product inhibition and equilibrium binding data suggest that MtPNP catalyzes 2'-deoxyguanosine (2dGuo) phosphorolysis by a steady-state ordered bi bi kinetic mechanism, in which inorganic phosphate (P(i)) binds first followed by 2dGuo, and ribose 1-phosphate dissociates first followed by guanine. pH-rate profiles indicated a general acid as being essential for both catalysis and 2dGuo binding, and that deprotonation of a group abolishes P(i) binding. Proton inventory and solvent deuterium isotope effects indicate that a single solvent proton transfer makes a modest contribution to the rate-limiting step. Pre-steady-state kinetic data indicate that product release appears to contribute to the rate-limiting step for MtPNP-catalyzed reaction.

  18. Capillary bioreactors based on human purine nucleoside phosphorylase: a new approach for ligands identification and characterization.

    PubMed

    de Moraes, Marcela Cristina; Ducati, Rodrigo Gay; Donato, Augusto José; Basso, Luiz Augusto; Santos, Diógenes Santiago; Cardoso, Carmen Lucia; Cass, Quezia Bezerra

    2012-04-01

    The enzyme purine nucleoside phosphorylase (PNP) is a target for the discovery of new lead compounds employed on the treatment severe T-cell mediated disorders. Within this context, the development of new, direct, and reliable methods for ligands screening is an important task. This paper describes the preparation of fused silica capillaries human PNP (HsPNP) immobilized enzyme reactor (IMER). The activity of the obtained IMER is monitored on line in a multidimensional liquid chromatography system, by the quantification of the product formed throughout the enzymatic reaction. The K(M) value for the immobilized enzyme was about twofold higher than that measured for the enzyme in solution (255 ± 29.2 μM and 133 ± 14.9 μM, respectively). A new fourth-generation immucillin derivative (DI4G; IC(50)=40.6 ± 0.36 nM), previously identified and characterized in HsPNP free enzyme assays, was used to validate the IMER as a screening method for HsPNP ligands. The validated method was also used for mechanistic studies with this inhibitor. This new approach is a valuable tool to PNP ligand screening, since it directly measures the hypoxanthine released by inosine phosphorolysis, thus furnishing more reliable results than those one used in a coupled enzymatic spectrophotometric assay.

  19. Structural studies of human purine nucleoside phosphorylase: towards a new specific empirical scoring function.

    PubMed

    Timmers, Luis Fernando Saraiva Macedo; Caceres, Rafael Andrade; Vivan, Ana Luiza; Gava, Lisandra Marques; Dias, Raquel; Ducati, Rodrigo Gay; Basso, Luiz Augusto; Santos, Diogenes Santiago; de Azevedo, Walter Filgueira

    2008-11-01

    Human purine nucleoside phosphorylase (HsPNP) is a target for inhibitor development aiming at T-cell immune response modulation. In this work, we report the development of a new set of empirical scoring functions and its application to evaluate binding affinities and docking results. To test these new functions, we solved the structure of HsPNP and 2-mercapto-4(3H)-quinazolinone (HsPNP:MQU) binary complex at 2.7A resolution using synchrotron radiation, and used these functions to predict ligand position obtained in docking simulations. We also employed molecular dynamics simulations to analyze HsPNP in two conditions, as apoenzyme and in the binary complex form, in order to assess the structural features responsible for stability. Analysis of the structural differences between systems provides explanation for inhibitor binding. The use of these scoring functions to evaluate binding affinities and molecular docking results may be used to guide future efforts on virtual screening focused on HsPNP.

  20. Insights into Brain Glycogen Metabolism: THE STRUCTURE OF HUMAN BRAIN GLYCOGEN PHOSPHORYLASE.

    PubMed

    Mathieu, Cécile; de la Sierra-Gallay, Ines Li; Duval, Romain; Xu, Ximing; Cocaign, Angélique; Léger, Thibaut; Woffendin, Gary; Camadro, Jean-Michel; Etchebest, Catherine; Haouz, Ahmed; Dupret, Jean-Marie; Rodrigues-Lima, Fernando

    2016-08-26

    Brain glycogen metabolism plays a critical role in major brain functions such as learning or memory consolidation. However, alteration of glycogen metabolism and glycogen accumulation in the brain contributes to neurodegeneration as observed in Lafora disease. Glycogen phosphorylase (GP), a key enzyme in glycogen metabolism, catalyzes the rate-limiting step of glycogen mobilization. Moreover, the allosteric regulation of the three GP isozymes (muscle, liver, and brain) by metabolites and phosphorylation, in response to hormonal signaling, fine-tunes glycogenolysis to fulfill energetic and metabolic requirements. Whereas the structures of muscle and liver GPs have been known for decades, the structure of brain GP (bGP) has remained elusive despite its critical role in brain glycogen metabolism. Here, we report the crystal structure of human bGP in complex with PEG 400 (2.5 Å) and in complex with its allosteric activator AMP (3.4 Å). These structures demonstrate that bGP has a closer structural relationship with muscle GP, which is also activated by AMP, contrary to liver GP, which is not. Importantly, despite the structural similarities between human bGP and the two other mammalian isozymes, the bGP structures reveal molecular features unique to the brain isozyme that provide a deeper understanding of the differences in the activation properties of these allosteric enzymes by the allosteric effector AMP. Overall, our study further supports that the distinct structural and regulatory properties of GP isozymes contribute to the different functions of muscle, liver, and brain glycogen.

  1. EXPRESSION PATTERNS OF THE GLYCOGEN PHOSPHORYLASE GENE RELATED TO LARVAL DIAPAUSE IN Ostrinia furnacalis.

    PubMed

    Guo, Jianqing; Zhang, Honggang; Edwards, Martin; Wang, Zhenying; Bai, Shuxiong; He, Kanglai

    2016-04-01

    Glycogen phosphorylase (GP) acts in the first step in release of glucose from glycogen, a form of energy storage for most organisms. To investigate the characteristics and expression pattern of GP gene (Ofgp) in the Asian corn borer, Ostrinia furnacalis (Guenée), larvae, we cloned and analyzed tissue transcription of Ofgp. The results indicate that the open reading frame (ORF) is 2,526 bp, encoding 841 amino acid. The calculated three-dimensional structure shows 33 α-helices and 24 β-sheets. Ofgp transcription levels varied significantly during the second to fifth instars under long-day (28 °C, 16:8 L:D photoperiod, and 70-80% relative humidity (RH)) and short-day (24.5 °C, 11:13 L:D photoperiod, and 70-80% RH) conditions, remained low during the prediapause phase, and then increased after about 36 d under short-day photoperiod. In the larvae reared under long-day condition, hemolymph ranked the highest in the transcript level of Ofgp. The highest transcription was recorded in the fat body and was lower in the other tissues in larvae reared under short-day condition. We found that Ofgp transcription increased linearly from October 2012 to January 2013. The transcript level was negatively correlated with environmental temperature. We infer the higher Ofgp transcription may enhance the cold hardiness of the diapause larvae. PMID:26748939

  2. Surface Induced Dissociation Yields Quaternary Substructure of Refractory Noncovalent Phosphorylase B and Glutamate Dehydrogenase Complexes

    NASA Astrophysics Data System (ADS)

    Ma, Xin; Zhou, Mowei; Wysocki, Vicki H.

    2014-03-01

    Ion mobility (IM) and tandem mass spectrometry (MS/MS) coupled with native MS are useful for studying noncovalent protein complexes. Collision induced dissociation (CID) is the most common MS/MS dissociation method. However, some protein complexes, including glycogen phosphorylase B kinase (PHB) and L-glutamate dehydrogenase (GDH) examined in this study, are resistant to dissociation by CID at the maximum collision energy available in the instrument. Surface induced dissociation (SID) was applied to dissociate the two refractory protein complexes. Different charge state precursor ions of the two complexes were examined by CID and SID. The PHB dimer was successfully dissociated to monomers and the GDH hexamer formed trimeric subcomplexes that are informative of its quaternary structure. The unfolding of the precursor and the percentages of the distinct products suggest that the dissociation pathways vary for different charge states. The precursors at lower charge states (+21 for PHB dimer and +27 for GDH hexamer) produce a higher percentage of folded fragments and dissociate more symmetrically than the precusors at higher charge states (+29 for PHB dimer and +39 for GDH hexamer). The precursors at lower charge state may be more native-like than the higher charge state because a higher percentage of folded fragments and a lower percentage of highly charged unfolded fragments are detected. The combination of SID and charge reduction is shown to be a powerful tool for quaternary structure analysis of refractory noncovalent protein complexes, as illustrated by the data for PHB dimer and GDH hexamer.

  3. Effects of thymidine phosphorylase on tumor aggressiveness and 5-fluorouracil sensitivity in cholangiocarcinoma

    PubMed Central

    Thanasai, Jongkonnee; Limpaiboon, Temduang; Jearanaikoon, Patcharee; Sripa, Banchob; Pairojkul, Chawalit; Tantimavanich, Srisurang; Miwa, Masanao

    2010-01-01

    AIM: To evaluate the role of thymidine phosphorylase (TP) in cholangiocarcinoma using small interfering RNA (siRNA). METHODS: A human cholangiocarcinoma-derived cell line KKU-M139, which has a naturally high level of endogenous TP, had TP expression transiently knocked down using siRNA. Cell growth, migration, in vitro angiogenesis, apoptosis, and cytotoxicity were assayed in TP knockdown and wild-type cell lines. RESULTS: TP mRNA and protein expression were decreased by 87.1% ± 0.49% and 72.5% ± 3.2%, respectively, compared with control cells. Inhibition of TP significantly decreased migration of KKU-M139, and suppressed migration and tube formation of human umbilical vein endothelial cells. siRNA also reduced the ability of TP to resist hypoxia-induced apoptosis, while suppression of TP reduced the sensitivity of KKU-M139 to 5-fluorouracil. CONCLUSION: Inhibition of TP may be beneficial in decreasing angiogenesis-dependent growth and migration of cholangiocarcinoma but may diminish the response to 5-fluorouracil chemotherapy. PMID:20355241

  4. Production and application of a rare disaccharide using sucrose phosphorylase from Leuconostoc mesenteroides.

    PubMed

    Morimoto, Kenji; Yoshihara, Akihide; Furumoto, Toshio; Takata, Goro

    2015-06-01

    Sucrose phosphorylase (SPase) from Leuconostoc mesenteroides exhibited activity towards eight ketohexoses, which behaved as D-glucosyl acceptors, and α-D-glucose-1-phosphate (G1P), which behaved as a donor. All eight of these ketohexoses were subsequently transformed into the corresponding d-glucosyl-ketohexoses. Of the eight ketohexoses evaluated in the current study, d-allulose behaved as the best substrate for SPase, and the resulting d-glucosyl-d-alluloside product was found to be a non-reducing sugar with a specific optical rotation of [α]D(20) + 74.36°. D-Glucosyl-D-alluloside was identified as α-D-glucopyranosyl-(1→2)-β-D-allulofuranoside by NMR analysis. D-Glucosyl-D-alluloside exhibited an inhibitory activity towards an invertase from yeast with a Km value of 50 mM, where it behaved as a competitive inhibitor with a Ki value of 9.2 mM. D-Glucosyl-D-alluloside was also successfully produced from sucrose using SPase and D-tagatose 3-epimerase. This process also allowed for the production of G1P from sucrose and d-allulose from D-fructose, which suggested that this method could be used to prepare d-glucosyl-d-alluloside without the need for expensive reagents such as G1P and d-allulose. PMID:25499751

  5. Physicochemical properties of quinoa starch.

    PubMed

    Li, Guantian; Wang, Sunan; Zhu, Fan

    2016-02-10

    Physicochemical properties of quinoa starches isolated from 26 commercial samples from a wide range of collection were studied. Swelling power (SP), water solubility index (WSI), amylose leaching (AML), enzyme susceptibility, pasting, thermal and textural properties were analyzed. Apparent amylose contents (AAM) ranged from 7.7 to 25.7%. Great variations in the diverse physicochemical properties were observed. Correlation analysis showed that AAM was the most significant factor related to AML, WSI, and pasting parameters. Correlations among diverse physicochemical parameters were analyzed. Principal component analysis using twenty three variables were used to visualize the difference among samples. Six principal components were extracted which could explain 88.8% of the total difference. The wide variations in physicochemical properties could contribute to innovative utilization of quinoa starch for food and non-food applications.

  6. Physicochemical properties of quinoa starch.

    PubMed

    Li, Guantian; Wang, Sunan; Zhu, Fan

    2016-02-10

    Physicochemical properties of quinoa starches isolated from 26 commercial samples from a wide range of collection were studied. Swelling power (SP), water solubility index (WSI), amylose leaching (AML), enzyme susceptibility, pasting, thermal and textural properties were analyzed. Apparent amylose contents (AAM) ranged from 7.7 to 25.7%. Great variations in the diverse physicochemical properties were observed. Correlation analysis showed that AAM was the most significant factor related to AML, WSI, and pasting parameters. Correlations among diverse physicochemical parameters were analyzed. Principal component analysis using twenty three variables were used to visualize the difference among samples. Six principal components were extracted which could explain 88.8% of the total difference. The wide variations in physicochemical properties could contribute to innovative utilization of quinoa starch for food and non-food applications. PMID:26686137

  7. Formation of nanoporous aerogels from wheat starch.

    PubMed

    Ubeyitogullari, Ali; Ciftci, Ozan N

    2016-08-20

    Biodegradable nanoporous aerogels were obtained from wheat starch using a simple and green method based on supercritical carbon dioxide (SC-CO2) drying. Effects of processing parameters (temperature, wheat starch concentration and mixing rate during gelatinization; temperature, pressure, and flow rate of CO2, during SC-CO2 drying) on the aerogel formation were investigated, and optimized for the highest surface area and smallest pore size of the aerogels. At the optimized conditions, wheat starch aerogels had surface areas between 52.6-59.7m(2)/g and densities ranging between 0.05-0.29g/cm(3). The average pore size of the starch aerogels was 20nm. Starch aerogels were stable up to 280°C. Due to high surface area and nanoporous structure, wheat starch aerogels are promising carrier systems for bioactives and drugs in food and pharmaceutical industries. PMID:27178916

  8. Biochemistry and genetics of starch synthesis.

    PubMed

    Keeling, Peter L; Myers, Alan M

    2010-01-01

    Enormous progress has been made in understanding the genetics and biochemistry of starch synthesis in crop plants. Furthermore, starch remains at the very epicenter of the world's food and feed chains and has even now become one of the world's most important sources of biorenewable energy (biofuel). Yet, despite this remarkable progress and the obvious economic importance, very little has been achieved in terms of adding value to starch or increasing starch yield, particularly in cereal crops. Here, we review the genetics and biochemistry of starch synthesis in crop plants, particularly maize. With all this know-how in place and a chasm of opportunity ahead, the time is right to see science deliver progress into a new frontier. Thus, in our view the stage is set for a new era of changes in starch synthesis, delivering enhancements in functionality and yield.

  9. The alpha and beta subunits of phosphorylase kinase are homologous: cDNA cloning and primary structure of the beta subunit.

    PubMed Central

    Kilimann, M W; Zander, N F; Kuhn, C C; Crabb, J W; Meyer, H E; Heilmeyer, L M

    1988-01-01

    We have cloned cDNA molecules encoding the beta subunit of phosphorylase kinase (ATP:phosphorylase-b phosphotransferase; EC 2.7.1.38) from rabbit fast-twitch skeletal muscle and have determined the complete primary structure of the polypeptide by a combination of peptide and DNA sequencing. In the mature beta subunit, the initial methionine is replaced by an acetyl group. The subunit is composed of 1092 amino acids and has a calculated molecular mass of 125,205 Da. Alignment of its sequence with the alpha subunit of phosphorylase kinase reveals extensive regions of homology, but each molecule also possesses unique sequences. Two of the three phosphorylation sites known for the beta subunit and all seven phosphorylation sites known for the alpha subunit are located in these unique domains. Images PMID:3200826

  10. Mutations in Durum Wheat SBEII Genes affect Grain Yield Components, Quality, and Fermentation Responses in Rats

    PubMed Central

    Hazard, Brittany; Zhang, Xiaoqin; Naemeh, Mahmoudreza; Hamilton, M. Kristina; Rust, Bret; Raybould, Helen E.; Newman, John W.; Martin, Roy; Dubcovsky, Jorge

    2016-01-01

    Increased amylose in wheat (Triticum ssp.) starch is associated with increased resistant starch, a fermentable dietary fiber. Fermentation of resistant starch in the large intestine produces short-chain fatty acids that are associated with human health benefits. Since wheat foods are an important component of the human diet, increases in amylose and resistant starch in wheat grains have the potential to deliver health benefits to a large number of people. In three replicated field trials we found that mutations in starch branching enzyme II genes (SBEIIa and SBEIIb) in both A and B genomes (SBEIIa/b-AB) of durum wheat [T. turgidum L. subsp. durum (Desf.) Husn.] resulted in large increases of amylose and resistant starch content. The presence of these four mutations was also associated with an average 5% reduction in kernel weight (P = 0.0007) and 15% reduction in grain yield (P = 0.06) compared to the wild type. Complete milling and pasta quality analysis showed that the mutant lines have an acceptable quality with positive effects on pasta firmness and negative effects on semolina extraction and pasta color. Positive fermentation responses were detected in rats (Rattus spp.) fed with diets incorporating mutant wheat flour. This study quantifies benefits and limitations associated with the deployment of the SBEIIa/b-AB mutations in durum wheat and provides the information required to develop realistic strategies to deploy durum wheat varieties with increased levels of amylose and resistant starch. PMID:27134286

  11. Structural and functional properties of C-type starches.

    PubMed

    Cai, Jinwen; Cai, Canhui; Man, Jianmin; Zhou, Weidong; Wei, Cunxu

    2014-01-30

    This study investigated the structural and functional properties of C-type starches from pea seeds, faba bean seeds, yam rhizomes and water chestnut corms. These starches were mostly oval in shape with significantly different sizes and contents of amylose, damaged starch and phosphorus. Pea, faba bean and water chestnut starches had central hila, and yam starch had eccentric hilum. Water chestnut and yam starches had higher amylopectin short and long chain, respectively. Water chestnut and faba bean starches showed CA-type crystallinities, and pea and yam starches had C-type crystallinities. Water chestnut starch had the highest swelling power, granule swelling and pasting viscosity, lowest gelatinization temperatures and enthalpy. Faba bean starch had the lowest pasting viscosity, whereas yam starch had the highest gelatinization temperatures. Water chestnut and yam starches possessed significantly higher and lower susceptibility to acid and enzyme hydrolysis, the highest and lowest RDS contents, and the lowest and highest RS contents, respectively.

  12. Starch aerogel beads obtained from inclusion complexes prepared from high amylose starch and sodium palmitate

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Starch aerogels are a class of low density highly porous renewable materials currently prepared from retrograded starch gels and are of interest for their good surface area, porosity, biocompatibility, and biodegradability. Recently, we have reported on starches containing amylose-fatty acid salt h...

  13. Starch-Soybean Oil Composites with High Oil: Starch Ratios Prepared by Steam Jet Cooking

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aqueous mixtures of soybean oil and starch were jet cooked at oil:starch ratios ranging from 0.5:1 to 4:1 to yield dispersions of micron-sized oil droplets that were coated with a thin layer of starch at the oil-water interface. The jet cooked dispersions were then centrifuged at 2060 and 10,800 x ...

  14. Inducing PLA/starch compatibility through butyl-etherification of waxy and high amylose starch.

    PubMed

    Wokadala, Obiro Cuthbert; Emmambux, Naushad Mohammad; Ray, Suprakas Sinha

    2014-11-01

    In this study, waxy and high amylose starches were modified through butyl-etherification to facilitate compatibility with polylactide (PLA). Fourier transform infrared spectroscopy, proton nuclear magnetic resonance spectroscopy and wettability tests showed that hydrophobic butyl-etherified waxy and high amylose starches were obtained with degree of substitution values of 2.0 and 2.1, respectively. Differential scanning calorimetry, tensile testing, and scanning electron microscopy (SEM) demonstrated improved PLA/starch compatibility for both waxy and high amylose starch after butyl-etherification. The PLA/butyl-etherified waxy and high amylose starch composite films had higher tensile strength and elongation at break compared to PLA/non-butyl-etherified composite films. The morphological study using SEM showed that PLA/butyl-etherified waxy starch composites had a more homogenous microstructure compared to PLA/butyl-etherified high amylose starch composites. Thermogravimetric analysis showed that PLA/starch composite thermal stability decreased with starch butyl-etherification for both waxy and high amylose starches. This study mainly demonstrates that PLA/starch compatibility can be improved through starch butyl-etherification.

  15. Inducing PLA/starch compatibility through butyl-etherification of waxy and high amylose starch.

    PubMed

    Wokadala, Obiro Cuthbert; Emmambux, Naushad Mohammad; Ray, Suprakas Sinha

    2014-11-01

    In this study, waxy and high amylose starches were modified through butyl-etherification to facilitate compatibility with polylactide (PLA). Fourier transform infrared spectroscopy, proton nuclear magnetic resonance spectroscopy and wettability tests showed that hydrophobic butyl-etherified waxy and high amylose starches were obtained with degree of substitution values of 2.0 and 2.1, respectively. Differential scanning calorimetry, tensile testing, and scanning electron microscopy (SEM) demonstrated improved PLA/starch compatibility for both waxy and high amylose starch after butyl-etherification. The PLA/butyl-etherified waxy and high amylose starch composite films had higher tensile strength and elongation at break compared to PLA/non-butyl-etherified composite films. The morphological study using SEM showed that PLA/butyl-etherified waxy starch composites had a more homogenous microstructure compared to PLA/butyl-etherified high amylose starch composites. Thermogravimetric analysis showed that PLA/starch composite thermal stability decreased with starch butyl-etherification for both waxy and high amylose starches. This study mainly demonstrates that PLA/starch compatibility can be improved through starch butyl-etherification. PMID:25129738

  16. Position of modifying groups on starch chains of octenylsuccinic anhydride-modified waxy maize starch.

    PubMed

    Bai, Yanjie; Kaufman, Rhett C; Wilson, Jeff D; Shi, Yong-Cheng

    2014-06-15

    Octenylsuccinic anhydride (OSA)-modified starches with a low (0.018) and high (0.092) degree of substitution (DS) were prepared from granular native waxy maize starch in aqueous slurry. The position of OS substituents along the starch chains was investigated by enzyme hydrolysis followed by chromatographic analysis. Native starch and two OS starches with a low and high DS had β-limit values of 55.9%, 52.8%, and 34.4%, respectively. The weight-average molecular weight of the β-limit dextrin from the OS starch with a low DS was close to that of the β-limit dextrin from native starch but lower than that of the β-limit dextrin from the OS starch with a high DS. Debranching of OS starches was incomplete compared with native starch. OS groups in the OS starch with a low DS were located on the repeat units near the branching points, whereas the OS substituents in the OS starch with a high DS occurred both near the branching points and the non-reducing ends.

  17. A novel GDP-D-glucose phosphorylase involved in quality control of the nucleoside diphosphate sugar pool in Caenorhabditis elegans and mammals.

    PubMed

    Adler, Lital N; Gomez, Tara A; Clarke, Steven G; Linster, Carole L

    2011-06-17

    The plant VTC2 gene encodes GDP-L-galactose phosphorylase, a rate-limiting enzyme in plant vitamin C biosynthesis. Genes encoding apparent orthologs of VTC2 exist in both mammals, which produce vitamin C by a distinct metabolic pathway, and in the nematode worm Caenorhabditis elegans where vitamin C biosynthesis has not been demonstrated. We have now expressed cDNAs of the human and worm VTC2 homolog genes (C15orf58 and C10F3.4, respectively) and found that the purified proteins also display GDP-hexose phosphorylase activity. However, as opposed to the plant enzyme, the major reaction catalyzed by these enzymes is the phosphorolysis of GDP-D-glucose to GDP and D-glucose 1-phosphate. We detected activities with similar substrate specificity in worm and mouse tissue extracts. The highest expression of GDP-D-glucose phosphorylase was found in the nervous and male reproductive systems. A C. elegans C10F3.4 deletion strain was found to totally lack GDP-D-glucose phosphorylase activity; this activity was also found to be decreased in human HEK293T cells transfected with siRNAs against the human C15orf58 gene. These observations confirm the identification of the worm C10F3.4 and the human C15orf58 gene expression products as the GDP-D-glucose phosphorylases of these organisms. Significantly, we found an accumulation of GDP-D-glucose in the C10F3.4 mutant worms, suggesting that the GDP-D-glucose phosphorylase may function to remove GDP-D-glucose formed by GDP-D-mannose pyrophosphorylase, an enzyme that has previously been shown to lack specificity for its physiological D-mannose 1-phosphate substrate. We propose that such removal may prevent the misincorporation of glucosyl residues for mannosyl residues into the glycoconjugates of worms and mammals.

  18. Photosynthate Partitioning into Starch in Soybean Leaves

    PubMed Central

    Chatterton, N. Jerry; Silvius, John E.

    1979-01-01

    Photosynthesis, photosynthate partitioning into foliar starch, and translocation were investigated in soybean plants (Glycine max (L.) Merr. cv. Amsoy 71), grown under different photoperiods and photosynthetic periods to determine the controls of leaf starch accumulation. Starch accumulation rates in soybean leaves were inversely related to the length of the daily photosynthetic period under which the plants were grown. Photosynthetic period and not photoperiod per se appears to be the important factor. Plants grown in a 14-hour photosynthetic period partitioned approximately 60% of the daily foliar accumulation into starch whereas 7-hour plants partitioned about 90% of their daily foliar accumulation into starch. The difference in starch accumulation resulted from a change in photosynthate partitioning between starch and leaf residual dry weight. Residual dry weight is defined as leaf dry weight minus the weight of total nonstructural carbohydrates. Differences in photosynthate partitioning into starch were also associated with changes in photosynthetic and translocation rates, as well as with leaf and whole plant morphology. It is concluded that leaf starch accumulation is a programmed process and not simply the result of a limitation in translocation. PMID:16661047

  19. Improved method for detection of starch hydrolysis

    SciTech Connect

    Ohawale, M.R.; Wilson, J.J.; Khachatourians, G.G.; Ingledew, W.M.

    1982-09-01

    A new starch hydrolysis detection method which does not rely on iodine staining or the use of color-complexed starch is described. A linear relationship was obtained with agar-starch plates when net clearing zones around colonies of yeasts were plotted against enzyme levels (semilogarithm scale) produced by the same yeast strains in liquid medium. A similar relationship between starch clearing zones and alpha-amylase levels from three different sources was observed. These observations suggest that the method is useful in mutant isolations, strain improvement programs, and the prediction of alpha-amylase activities in culture filtrates or column effluents. (Refs. 18).

  20. Physical modification of food starch functionalities.

    PubMed

    BeMiller, James N; Huber, Kerry C

    2015-01-01

    Because, in general, native starches do not have properties that make them ideally suited for applications in food products, most starch is modified by dervatization to improve its functionality before use in processed food formulations, and because food processors would prefer not to have to use the modified food starch label designation required when chemically modified starches are used, there is considerable interest in providing starches with desired functionalities that have not been chemically modified. One investigated approach is property modification via physical treatments, that is, modifications of starches imparted by physical treatments that do not result in any chemical modification of the starch. Physical treatments are divided into thermal and nonthermal treatments. Thermal treatments include those that produce pregelatinized and granular cold-water-swelling starches, heat-moisture treatments, annealing, microwave heating, so-called osmotic pressure treatment, and heating of dry starch. Nonthermal treatments include ultrahigh-pressure treatments, instantaneous controlled pressure drop, use of high-pressure homogenizers, dynamic pulsed pressure, pulsed electric field, and freezing and thawing.

  1. Reduced gravitropism in hypocotyls of starch-deficient mutants of Arabidopsis

    NASA Technical Reports Server (NTRS)

    Kiss, J. Z.; Guisinger, M. M.; Miller, A. J.; Stackhouse, K. S.

    1997-01-01

    Gravitropism was examined in dark- and light-grown hypocotyls of wild-type (WT), two reduced starch mutants (ACG 20 and ACG 27), and a starchless mutant (ACG 21) of Arabidopsis. In addition, the starch content of these four strains was studied with light and electron microscopy. Based on time course of curvature and orientation studies, the graviresponse in hypocotyls is proportional to the amount of starch in a genotype. Furthermore, starch mutations seem to primarily affect gravitropism rather than differential growth since both phototropic curvature and growth rates among the four genotypes are approximately equal. Our results suggest that gravity perception may require a greater plastid mass in hypocotyls compared to roots. The kinetics of gravitropic curvature also was compared following reorientation at 45 degrees, 90 degrees, and 135 degrees. As has been reported for other plant species, the optimal angle of reorientation is 135 degrees for WT Arabidopsis and the two reduced starch mutants, but the magnitude of curvature of the starchless mutant appears to be independent of the initial angle of displacement. Taken together, the results of the present study and our previous experiments with roots of the same four genotypes [Kiss et al. (1996) Physiol. Plant. 97: 237] support a plastid-based hypothesis for gravity perception in plants.

  2. The phosphate site of trehalose phosphorylase from Schizophyllum commune probed by site-directed mutagenesis and chemical rescue studies.

    PubMed

    Goedl, Christiane; Nidetzky, Bernd

    2008-03-01

    Schizophyllum communealpha,alpha-trehalose phosphorylase utilizes a glycosyltransferase-like catalytic mechanism to convert its disaccharide substrate into alpha-d-glucose 1-phosphate and alpha-d-glucose. Recruitment of phosphate by the free enzyme induces alpha,alpha-trehalose binding recognition and promotes the catalytic steps. Like the structurally related glycogen phosphorylase and other retaining glycosyltransferases of fold family GT-B, the trehalose phosphorylase contains an Arg507-XXXX-Lys512 consensus motif (where X is any amino acid) comprising key residues of its putative phosphate-binding sub-site. Loss of wild-type catalytic efficiency for reaction with phosphate (kcat/Km=21,000 m(-1).s(-1)) was dramatic (>or=10(7)-fold) in purified Arg507-->Ala (R507A) and Lys512-->Ala (K512A) enzymes, reflecting a corresponding change of comparable magnitude in kcat (Arg507) and Km (Lys512). External amine and guanidine derivatives selectively enhanced the activity of the K512A mutant and the R507A mutant respectively. Analysis of the pH dependence of chemical rescue of the K512A mutant by propargylamine suggested that unprotonated amine in combination with H2PO4-, the protonic form of phosphate presumably utilized in enzymatic catalysis, caused restoration of activity. Transition state-like inhibition of the wild-type enzyme A by vanadate in combination with alpha,alpha-trehalose (Ki=0.4 microm) was completely disrupted in the R507A mutant but only weakened in the K512A mutant (Ki=300 microm). Phosphate (50 mm) enhanced the basal hydrolase activity of the K512A mutant toward alpha,alpha-trehalose by 60% but caused its total suppression in wild-type and R507A enzymes. The results portray differential roles for the side chains of Lys512 and Arg507 in trehalose phosphorylase catalysis, reactant state binding of phosphate and selective stabilization of the transition state respectively. PMID:18205830

  3. Four Generations of Transition State Analogues for Human Purine Nucleoside Phosphorylase

    SciTech Connect

    Ho, M.; Shi, W; Rinaldo-Mathis, A; Tyler, P; Evans, G; Almo, S; Schramm, V

    2010-01-01

    Inhibition of human purine nucleoside phosphorylase (PNP) stops growth of activated T-cells and the formation of 6-oxypurine bases, making it a target for leukemia, autoimmune disorders, and gout. Four generations of ribocation transition-state mimics bound to PNP are structurally characterized. Immucillin-H (K*{sub i} = 58 pM, first-generation) contains an iminoribitol cation with four asymmetric carbons. DADMe-Immucillin-H (K*{sub i} = 9 pM, second-generation), uses a methylene-bridged dihydroxypyrrolidine cation with two asymmetric centers. DATMe-Immucillin-H (K*{sub i} = 9 pM, third-generation) contains an open-chain amino alcohol cation with two asymmetric carbons. SerMe-ImmH (K*{sub i} = 5 pM, fourth-generation) uses achiral dihydroxyaminoalcohol seramide as the ribocation mimic. Crystal structures of PNPs establish features of tight binding to be; (1) ion-pair formation between bound phosphate (or its mimic) and inhibitor cation, (2) leaving-group interactions to N1, O6, and N7 of 9-deazahypoxanthine, (3) interaction between phosphate and inhibitor hydroxyl groups, and (4) His257 interacting with the 5{prime}-hydroxyl group. The first generation analogue is an imperfect fit to the catalytic site with a long ion pair distance between the iminoribitol and bound phosphate and weaker interactions to the leaving group. Increasing the ribocation to leaving-group distance in the second- to fourth-generation analogues provides powerful binding interactions and a facile synthetic route to powerful inhibitors. Despite chemical diversity in the four generations of transition-state analogues, the catalytic site geometry is almost the same for all analogues. Multiple solutions in transition-state analogue design are available to convert the energy of catalytic rate enhancement to binding energy in human PNP.

  4. Conformational States of Human Purine Nucleoside Phosphorylase at Rest, at Work and with Transition State Analogues†

    PubMed Central

    Edwards, Achelle A.; Tipton, Jeremiah D.; Brenowitz, Michael D.; Emmett, Mark R.; Marshall, Alan G.; Evans, Gary B.; Tyler, Peter C.; Schramm, Vern L.

    2010-01-01

    Human purine nucleoside phosphorylase (PNP) is a homotrimer binding tightly to the transition state analogues Immucillin-H (ImmH, Kd = 56 pM) and DATMe-ImmH-Immucillin-H (DATMe-ImmH, Kd = 8.6 pM). ImmH binds with a larger entropic penalty than DATMe-ImmH, a chemically more flexible inhibitor. The testable hypothesis is that PNP conformational states are more relaxed (dynamic) with DATMe-ImmH, despite tighter binding than with ImmH. PNP conformations are probed by peptide amide deuterium exchange (HDX) using liquid chromatography high-resolution Fourier transform ion cyclotron resonance mass spectrometry and by sedimentation rates. Catalytically equilibrating Michaelis complexes (PNP•PO4•Inosine ↔ PNP•Hx•R-1-P) and inhibited complexes (PNP•PO4•DATMe-ImmH and PNP•PO4•ImmH) show protection from HDX at 9, 13 and 15 sites per subunit relative to resting PNP (PNP•PO4) in extended incubations. The PNP•PO4•ImmH complex is more compact (by sedimentation rate) than the other complexes. HDX kinetic analysis of ligand-protected sites corresponds to peptides near the catalytic sites. HDX and sedimentation results establish that PNP protein conformation (dynamic motion) correlates more closely to entropy of binding than to affinity. Catalytically active turnover with saturated substrate sites causes less change in HDX and sedimentation rates than binding of transition state analogues. DATMe-ImmH more closely mimics the transition of human PNP than does ImmH, and achieves strong binding interactions at the catalytic site while causing relatively modest alterations of the protein dynamic motion. Transition state analogues causing the most rigid, closed protein conformation are therefore not necessarily the most tightly bound. Close mimics of the transition state are hypothesized to retain enzymatic dynamic motions related to transition state formation. PMID:20108972

  5. Thymidine phosphorylase induces angiogenesis in vivo and in vitro: an evaluation of possible mechanisms

    PubMed Central

    Sengupta, Shiladitya; Sellers, Lynda A; Matheson, Hugh B; Fan, Tai-Ping D

    2003-01-01

    Thymidine phosphorylase (TP) is elevated in the plasma of cancer patients, and has been implicated in pathophysiological angiogenesis. However, the downstream signals underlying this implication remain obscure. The purpose of the present study was to examine the effects of TP on the neovascularisation response in vitro and in vivo. Both TP and its catalytic product, 2-deoxy-D-ribose-1-phosphate, and downstream 2-deoxy-D-ribose (2-DDR) promoted endothelial tubulogenesis in vitro, and the regeneration of a wounded monolayer of endothelial cells without exerting any mitogenic effect. In vivo, both TP and 2-DDR promoted the development of functional vasculature into an avascular sponge. A TP inhibitor, 6-amino-5-chlorouracil, was able to partially reverse the effects of TP, but had no effect on the 2-DDR-induced angiogenesis. Enhanced monolayer regeneration was observed with TP-cDNA-transfected bladder carcinoma cells. The transfection of TP-cDNA, however, did not confer any proliferative advantage. The regeneration of TP overexpressing cells was associated with a time-dependent expression of the enzyme haeme-oxygenase (HO-1). The present study demonstrates that both TP and its ribose-sugar metabolites induce angiogenesis by mediating a cohesive interplay between carcinoma and endothelial cells. The induction of HO-1 in TP-transfected cells suggests that it could be a possible downstream signal for the angiogenic effects of TP. Furthermore, reducing sugars have been shown to induce oxidative stress, and ribose could be a possible cause for the upregulation of HO-1, which has been implicated in the release of angiogenic factors. Therefore, we postulate that 2-DDR could be mediating the angiogenic effects of TP possibly through an oxidative stress mechanism and additionally getting integrated in the endothelial metabolic machinery. PMID:12770927

  6. Transition Path Sampling Study of the Reaction Catalyzed by Purine Nucleoside Phosphorylase

    PubMed Central

    Saen-oon, Suwipa; Schramm, Vern L.; Schwartz, Steven D.

    2010-01-01

    The Transition Path Sampling (TPS) method is a powerful technique for studying rare events in complex systems, that allows description of reactive events in atomic detail without prior knowledge of reaction coordinates and transition states. We have applied TPS in combination with a hybrid Quantum Mechanical/Molecular Mechanical (QM/MM) method to study the enzyme human purine nucleoside phosphorylase (hPNP). This enzyme catalyzes the reversible phosphorolysis of 6-oxypurine (deoxy)nucleosides to generate the corresponding purine base and (deoxy)ribose 1-phosphate. Hundreds of reactive trajectories were generated. Analysis of this transition path ensembles provides insight into the detailed mechanistic dynamics of reaction in the enzyme. Our studies have indicated a reaction mechanism involving the cleavage of the N-ribosidic bond to form transition states with substantial ribooxacarbenium ion character, that is then followed by conformational changes in the enzyme and the ribosyl group leading to migration of the anomeric carbon of the ribosyl group toward phosphate to form the product ribose 1-phosphate. This latter process is crucial in PNP, because several strong H-bonds form between active site residues in order to capture and align the phosphate nucleophile. Calculations of the commitment probability along reactive paths demonstrated the presence of a broad energy barrier at the transition state. Analysis of these transition state structures showed that bond-breaking and bond-forming distances are not a good choice for the reaction coordinate, but that the pseudorotational phase of the ribose ring is also a significant variable. PMID:20664707

  7. Characterization and Prognostic Significance of Methylthioadenosine Phosphorylase Deficiency in Nasopharyngeal Carcinoma

    PubMed Central

    He, Hong-Lin; Lee, Ying-En; Shiue, Yow-Ling; Lee, Sung-Wei; Chen, Tzu-Ju; Li, Chien-Feng

    2015-01-01

    Abstract Identification of cancer-associated genes by genomic profiling contributes to the elucidation of tumor development and progression. The methylthioadenosine phosphorylase (MTAP) gene, located at chromosome 9p21, plays a critical role in tumorigenicity and disease progression in a wide variety of cancers. However, the prognostic impact of MTAP in patients with nasopharyngeal carcinoma (NPC) remains obscured. Through data mining from published transcriptomic database, MTAP was first identified as a differentially downregulated gene in NPC. In this study, our aim was to evaluate the expression of MTAP in NPC and to clarify its prognostic significance. MTAP immunohistochemistry was retrospectively performed and analyzed in biopsy specimens from 124 NPC patients who received standard treatment without distant metastasis at initial diagnosis. The immunoexpression status was correlated with the clinicopathological variables, disease-specific survival (DSS), distant metastasis-free survival (DMFS), and local recurrence-free survival (LRFS). Real-time quantitative polymerase chain reaction (PCR) was used to measure MTAP gene dosage. In some cases, we also performed methylation-specific PCR and pyrosequencing to assess the status of promoter methylation. MTAP deficiency was significantly associated with advanced tumor stages (P = 0.023) and univariately predictive of adverse outcomes for DSS, DMFS, and LRFS. In the multivariate comparison, MTAP deficiency still remained prognostically independent to portend worse DSS (P = 0.021, hazard ratio = 1.870) and DMFS (P = 0.009, hazard ratio = 2.154), together with advanced AJCC stages III to IV. Homozygous deletion or promoter methylation of MTAP gene were identified to be significantly associated with MTAP protein deficiency (P < 0.001). MTAP deficiency was correlated with an aggressive phenotype and independently predictive of worse DSS and DMFS, suggesting its role in disease progression and as an

  8. Cryoelectron microscopy reveals new features in the three-dimensional structure of phosphorylase kinase.

    PubMed

    Nadeau, Owen W; Gogol, Edward P; Carlson, Gerald M

    2005-04-01

    Phosphorylase kinase (PhK), a regulatory enzyme in the cascade activation of glycogenolysis, is a 1.3-MDa hexadecameric complex, (alphabetagammadelta)(4). PhK comprises two arched octameric (alphabetagammadelta)(2) lobes that are oriented back-to-back with overall D(2) symmetry and connected by small bridges. These interlobal bridges, arguably the most questionable structural component of PhK, are one of several structural features that potentially are artifactually generated or altered by conventional sample preparation techniques for electron microscopy (EM). To minimize such artifacts, we have solved by cryoEM the first three-dimensional (3D) structure of nonactivated PhK from images of frozen hydrated molecules of the kinase. Minimal dose electron micrographs of PhK in vitreous ice revealed particles in a multitude of orientations. A simple model was used to orient the individual images for 3D reconstruction, followed by multiple rounds of refinement. Three-dimensional reconstruction of nonactivated PhK from approximately 5000 particles revealed a bridged, bilobal molecule with a resolution estimated by Fourier shell correlation analysis at 25 A. This new structure suggests that several prominent features observed in the structure of PhK derived from negatively stained particles arise as artifacts of specimen preparation. In comparison to the structure from negative staining, the cryoEM structure shows three important differences: (1) a dihedral angle between the two lobes of approximately 90 degrees instead of 68 degrees, (2) a compact rather than extended structure for the lobes, and (3) the presence of four, rather than two, connecting bridges, which provides the first direct evidence for these components as authentic elements of the kinase solution structure. PMID:15741332

  9. Regulation of the Dictyostelium glycogen phosphorylase 2 gene by cyclic AMP.

    PubMed

    Sucic, J F; Selmin, O; Rutherford, C L

    1993-01-01

    A crucial developmental event in the cellular slime mold, Dictyostelium discoideum, is glycogen degradation. The enzyme that catalyzes this degradation, glycogen phosphorylase 2 (gp-2), is developmentally regulated and cAMP appears to be involved in this regulation. We have examined several aspects of the cAMP regulation of gp-2. We show that addition of exogenous cAMP to aggregation competent amoebae induced the appearance of gp-2 mRNA. The induction of gp-2 mRNA occurred within 1 and 1.5 h after the initial exposure to cAMP. Exposure to exogenous cAMP concentrations as low as 1.0 microM could induce gp-2 mRNA. We also examined the molecular mechanism through which cAMP induction of gp-2 occurs. Induction of gp-2 appears to result from a mechanism that does not require intracellular cAMP signaling, and may occur directly through a cAMP binding protein without the requirement of any intracellular signalling. We also examined the promoter region of the gp-2 gene for cis-acting elements that are involved in the cAMP regulation of gp-2. A series of deletions of the promoter were fused to a luciferase reporter gene and then analyzed for cAMP responsiveness. The results indicated that a region from -258 nucleotides to the transcriptional start site is sufficient for essentially full activity and appears to carry all necessary cis-acting sites for cAMP induction. Further deletion of 58 nucleotides from the 5' end, results in fivefold less activity in the presence of cAMP. Deletion of the next 104 nucleotides eliminates the cAMP response entirely. PMID:8222346

  10. Starch columns: Analog model for basalt columns

    NASA Astrophysics Data System (ADS)

    Müller, Gerhard

    1998-07-01

    Desiccation of starch-water mixtures produces tensile-crack patterns which appear to be interesting, but largely unknown study objects for fracture mechanics, structural geology, and volcanology. This paper concentrates on columnar jointing and on columns in starch. Starch columns have polygonal cross sections and are very similar to basalt columns. They are produced by lamp drying starch specimens with dimensions of several centimeters and have diameters in the millimeter range. The columns develop behind a crack front which propagates from the surface into the interior. The experiments, supported by X ray tomograms, show that polygonal regularity of the crack pattern is not present at the surface but develops during penetration. This transition is steered by a minimum-fracture-energy principle. The analogy between basalt cooling and starch desiccation is far reaching: water concentration in starch is analogous to temperature in basalt, both quantities obey diffusion equations, water loss is equivalent to heat loss, the resulting contraction stresses have similar dependences on depth and time, and in both cases the material strength is exceeded. The starch experiments show that column diameters are controlled by the depth gradient of water concentration at the crack front. High (low) gradients are connected with thin (thick) columns. By analogy, a similar relation with the temperature gradient exists for basalt columns. The (normalized) starch gradients are about 3 orders of magnitude larger than the (normalized) gradients in basalt. This explains why starch columns are much thinner than basalt columns. The gradients are so different, because the crack front speeds differ by a factor of about 10: after 3 days the speed is about 10 mm/d in starch but about 100 mm/d in basalt [Peck, 1978]. The speed difference, in turn, results from the difference of the diffusion constants: the hydraulic diffusivity of starch is 2 orders of magnitude lower than the thermal

  11. Characterisation of corn starch-based films reinforced with taro starch nanoparticles.

    PubMed

    Dai, Lei; Qiu, Chao; Xiong, Liu; Sun, Qingjie

    2015-05-01

    Taro starch nanoparticles (TSNPs) obtained by hydrolysis with pullulanase and the recrystallisation of gelatinised starch were used as reinforcing agents in corn starch films. The influence of TSNPs contents (0.5-15%) on the physical, mechanical, thermal, and structural properties of starch films was investigated. An increase in the concentration of TSNPs led to a significant decrease in the water vapour permeability (WVP) of films. The addition of TSNPs increased the tensile strength (TS) of films from 1.11 MPa to 2.87 MPa. Compared with pure starch films, the surfaces of nanocomposite films became uneven. The onset temperature (To) and melting temperature (Tm) of films containing TSNPs were higher than those of pure starch films. The addition of TSNPs improved the thermal stability of starch films. PMID:25529655

  12. Characterisation of corn starch-based films reinforced with taro starch nanoparticles.

    PubMed

    Dai, Lei; Qiu, Chao; Xiong, Liu; Sun, Qingjie

    2015-05-01

    Taro starch nanoparticles (TSNPs) obtained by hydrolysis with pullulanase and the recrystallisation of gelatinised starch were used as reinforcing agents in corn starch films. The influence of TSNPs contents (0.5-15%) on the physical, mechanical, thermal, and structural properties of starch films was investigated. An increase in the concentration of TSNPs led to a significant decrease in the water vapour permeability (WVP) of films. The addition of TSNPs increased the tensile strength (TS) of films from 1.11 MPa to 2.87 MPa. Compared with pure starch films, the surfaces of nanocomposite films became uneven. The onset temperature (To) and melting temperature (Tm) of films containing TSNPs were higher than those of pure starch films. The addition of TSNPs improved the thermal stability of starch films.

  13. Acetylation and characterization of banana (Musa paradisiaca) starch.

    PubMed

    Bello-Pérez, L A; Contreras-Ramos, S M; Jìmenez-Aparicio, A; Paredes-López, O

    2000-01-01

    Banana native starch was acetylated and some of its functional properties were evaluated and compared to corn starch. In general, acetylated banana starch presented higher values in ash, protein and fat than corn acetylated starch. The modified starches had minor tendency to retrogradation assessed as % transmittance of starch pastes. At high temperature acetylated starches presented a water retention capacity similar to their native counterpart. The acetylation considerably increased the solubility of starches, and a similar behavior was found for swelling power. When freeze-thaw stability was studied, acetyl banana starch drained approximately 60% of water in the first and second cycles, but in the third and fourth cycles the percentage of separated water was low. However, acetyl corn starch showed lower freeze-thaw stability than the untreated sample. The modification increased the viscosity of banana starch pastes.

  14. Functional properties of yam bean (Pachyrhizus erosus) starch.

    PubMed

    Mélo, E A; Stamford, T L M; Silva, M P C; Krieger, N; Stamford, N P

    2003-08-01

    The study was carried out in order to determine and establish the functional characters of starch extracted from yam bean (Pachyrhizus erosus (L) Urban) compared with cassava starch. Yam bean is a tropical tuber legume easily grown and holds a great potential as a new source of starch. Yam bean starch shows functional properties which are peculiar to those of most starch root crops. Gelatinization temperature (53-63 degrees C) and the pasting temperature (64.5 degrees C) are less than those of cereal starch, however, the swelling power is high (54.4 g gel/g dried starch). Yam bean starch paste presents a high viscosity profile, high retrogradation tendency and low stability on cooking. The functional properties of yam bean starch, similar to those of cassava starch, allows yam bean to be used as a potential new source of starch. PMID:12676508

  15. Sources and intake of resistant starch in the Chinese diet.

    PubMed

    Chen, Liyong; Liu, Ruiping; Qin, Chengyong; Meng, Yan; Zhang, Jie; Wang, Yun; Xu, Guifa

    2010-01-01

    Resistant starch (RS) escapes digestion in the small intestine and may ferment in the large intestine. The purpose of this study was to determine the resistant starch content in typical starchy foods and to estimate the daily resistant starch intake and identify key sources of dietary resistant starch in the Chinese diets. The resistant starch contents of 121 foods were determined using a method that mimicked gastrointestinal conditions. Tubers and legumes had high resistant starch contents. Rough food processing retained large amounts of resistant starch. In general, the content of RS decreased when foods were cooked. Deep fried and roasted foods had higher levels of resistant starch than braised foods. The average resistant starch intake in the Chinese population was estimated to be 14.9 g per day based on a dietary survey. The main resistant starch sources in the Chinese diet were cereal and tuber products. Based on dietary habits, however, the resistant starch intake varies considerably among individuals.

  16. Molecular Basis of the Waxy Endosperm Starch Phenotype in Broomcorn Millet (Panicum miliaceum L.)

    PubMed Central

    Hunt, Harriet V.; Denyer, Kay; Packman, Len C.; Jones, Martin K.; Howe, Christopher J.

    2010-01-01

    Waxy varieties of the tetraploid cereal broomcorn millet (Panicum miliaceum L.) have endosperm starch granules lacking detectable amylose. This study investigated the basis of this phenotype using molecular and biochemical methods. Iodine staining of starch granules in 72 plants from 38 landrace accessions found 58 nonwaxy and 14 waxy phenotype plants. All waxy types were in plants from Chinese and Korean accessions, a distribution similar to that of the waxy phenotype in other cereals. Granule-bound starch synthase I (GBSSI) protein was present in the endosperm of both nonwaxy and waxy individuals, but waxy types had little or no granule-bound starch synthase activity compared with the wild types. Sequencing of the GBSSI (Waxy) gene showed that this gene is present in two different forms (L and S) in P. miliaceum, which probably represent homeologues derived from two distinct diploid ancestors. Protein products of both these forms are present in starch granules. We identified three polymorphisms in the exon sequence coding for mature GBSSI peptides. A 15-bp deletion has occurred in the S type GBSSI, resulting in the loss of five amino acids from glucosyl transferase domain 1 (GTD1). The second GBSSI type (L) shows two sequence polymorphisms. One is the insertion of an adenine residue that causes a reading frameshift, and the second causes a cysteine–tyrosine amino acid polymorphism. These mutations appear to have occurred in parallel from the ancestral allele, resulting in three GBSSI-L alleles in total. Five of the six possible genotype combinations of the S and L alleles were observed. The deletion in the GBSSI-S gene causes loss of protein activity, and there was 100% correspondence between this deletion and the waxy phenotype. The frameshift mutation in the L gene results in the loss of L-type protein from starch granules. The L isoform with the tyrosine residue is present in starch granules but is nonfunctional. This loss of function may result from the

  17. Antimicrobial nanostructured starch based films for packaging.

    PubMed

    Abreu, Ana S; Oliveira, M; de Sá, Arsénio; Rodrigues, Rui M; Cerqueira, Miguel A; Vicente, António A; Machado, A V

    2015-09-20

    Montmorillonite modified with a quaternary ammonium salt C30B/starch nanocomposite (C30B/ST-NC), silver nanoparticles/starch nanocomposite (Ag-NPs/ST-NC) and both silver nanoparticles/C30B/starch nanocomposites (Ag-NPs/C30B/ST-NC) films were produced. The nanoclay (C30B) was dispersed in a starch solution using an ultrasonic probe. Different concentrations of Ag-NPs (0.3, 0.5, 0.8 and 1.0mM) were synthesized directly in starch and in clay/starch solutions via chemical reduction method. Dispersion of C30B silicate layers and Ag-NPs in ST films characterized by X-ray and scanning electron microscopy showed that the presence of Ag-NPs enhanced clay dispersion. Color and opacity measurements, barrier properties (water vapor and oxygen permeabilities), dynamic mechanical analysis and contact angle were evaluated and related with the incorporation of C30B and Ag-NPs. Films presented antimicrobial activity against Staphylococcus aureus, Escherichia coli and Candida albicans without significant differences between Ag-NPs concentrations. The migration of components from the nanostructured starch films, assessed by food contact tests, was minor and under the legal limits. These results indicated that the starch films incorporated with C30B and Ag-NPs have potential to be used as packaging nanostructured material. PMID:26050897

  18. Production of PLA-Starch Fibers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Composites of polylactic acid (PLA) with starch have been prepared previously in an effort to reduce cost as well as to modify other properties such as biodegradation rate. However, strength and elongation both decrease on addition of starch due to poor adhesion and stress concentration at the inte...

  19. Friction Properties of Chemically Modified Starch

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Starch is a high molecular weight polyglucose biopolymer that, in its native state, is insoluble in water at room temperature. One way of improving its water solubility is by esterification of its free hydroxyl groups. Waxy maize, normal corn, and high amylose corn starches were esterified with ac...

  20. Antimicrobial nanostructured starch based films for packaging.

    PubMed

    Abreu, Ana S; Oliveira, M; de Sá, Arsénio; Rodrigues, Rui M; Cerqueira, Miguel A; Vicente, António A; Machado, A V

    2015-09-20

    Montmorillonite modified with a quaternary ammonium salt C30B/starch nanocomposite (C30B/ST-NC), silver nanoparticles/starch nanocomposite (Ag-NPs/ST-NC) and both silver nanoparticles/C30B/starch nanocomposites (Ag-NPs/C30B/ST-NC) films were produced. The nanoclay (C30B) was dispersed in a starch solution using an ultrasonic probe. Different concentrations of Ag-NPs (0.3, 0.5, 0.8 and 1.0mM) were synthesized directly in starch and in clay/starch solutions via chemical reduction method. Dispersion of C30B silicate layers and Ag-NPs in ST films characterized by X-ray and scanning electron microscopy showed that the presence of Ag-NPs enhanced clay dispersion. Color and opacity measurements, barrier properties (water vapor and oxygen permeabilities), dynamic mechanical analysis and contact angle were evaluated and related with the incorporation of C30B and Ag-NPs. Films presented antimicrobial activity against Staphylococcus aureus, Escherichia coli and Candida albicans without significant differences between Ag-NPs concentrations. The migration of components from the nanostructured starch films, assessed by food contact tests, was minor and under the legal limits. These results indicated that the starch films incorporated with C30B and Ag-NPs have potential to be used as packaging nanostructured material.

  1. Pasting characteristics of starch-lipid composites

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Starch-lipid composites (SLC) have been used as fat replacers and stabilizers in beef patties, dairy products, and baked goods. The SLC are produced by mixing aqueous starch slurry with a lipid source, and steam jet-cooking. The SLC may be dried using a drum drier and then milled in a Retch mill. ...

  2. Preparation and characterization of octenylsuccinylated plantain starch.

    PubMed

    Bello-Flores, Christopher A; Nuñez-Santiago, Maria C; San Martín-Gonzalez, María F; BeMiller, James N; Bello-Pérez, Luis A

    2014-09-01

    Plantain starch was esterified with octenylsuccinic anhydride (OSA) at two concentrations (3 and 15% w/w) of OSA. The morphology, granule size distribution, pasting, gelatinization, swelling, and solubility of granules and structural features of the starch polymers were evaluated. Granules of the OSA-modified starches increased in size during cooking more than did the granules of the native starch, and the effect was greater at the higher OSA concentration. Pasting viscosities also increased, but gelatinization and pasting temperatures and enthalpy of gelatinization decreased in the OSA-modified starches. It was concluded that insertion of OS groups effected disorder in the granular structure. Solubility, weight average molar mass, Mw¯, and z-average radius of gyration, RGz, of the amylopectin decreased as the OSA concentration increased, indicating a decrease in molecular size.

  3. Preparation and characterization of octenylsuccinylated plantain starch.

    PubMed

    Bello-Flores, Christopher A; Nuñez-Santiago, Maria C; San Martín-Gonzalez, María F; BeMiller, James N; Bello-Pérez, Luis A

    2014-09-01

    Plantain starch was esterified with octenylsuccinic anhydride (OSA) at two concentrations (3 and 15% w/w) of OSA. The morphology, granule size distribution, pasting, gelatinization, swelling, and solubility of granules and structural features of the starch polymers were evaluated. Granules of the OSA-modified starches increased in size during cooking more than did the granules of the native starch, and the effect was greater at the higher OSA concentration. Pasting viscosities also increased, but gelatinization and pasting temperatures and enthalpy of gelatinization decreased in the OSA-modified starches. It was concluded that insertion of OS groups effected disorder in the granular structure. Solubility, weight average molar mass, Mw¯, and z-average radius of gyration, RGz, of the amylopectin decreased as the OSA concentration increased, indicating a decrease in molecular size. PMID:25036604

  4. Understanding starch gelatinization: The phase diagram approach.

    PubMed

    Carlstedt, Jonas; Wojtasz, Joanna; Fyhr, Peter; Kocherbitov, Vitaly

    2015-09-20

    By constructing a detailed phase diagram for the potato starch-water system based on data from optical microscopy, synchrotron X-ray scattering and differential scanning calorimetry, we show that gelatinization can be interpreted in analogy with a eutectic transition. The phase rule explains why the temperature of the gelatinization transition (G) is independent on water content. Furthermore, the melting (M1) endotherm observed in DSC represents a liquidus line; the temperature for this event increases with increasing starch concentration. Both the lamellar spacing and the inter-helix distance were observed to decrease with increasing starch content for starch concentrations between approximately 65 wt% and 75 wt%, while the inter-helix distance continued decreasing upon further dehydration. Understanding starch gelatinization has been a longstanding challenge. The novel approach presented here shows interpretation of this phenomenon from a phase equilibria perspective.

  5. Interaction of tannins and other sorghum phenolic compounds with starch and effects on in vitro starch digestibility.

    PubMed

    Barros, Frederico; Awika, Joseph M; Rooney, Lloyd W

    2012-11-21

    This study investigated interactions of sorghum proanthocyanidins (PAs) with starch molecules and the effect on in vitro starch digestibility. High tannin (predominant in PA), black (monomeric polyphenols), and white (low in polyphenols) sorghum phenolic extracts were mixed and cooked with starches varying in amylose content. Starch pasting properties, polyphenol profile, and resistant starch (RS) were determined. PAs decreased setback of normal starch and were least extractable after cooking with all starches. Pure amylose interacted more strongly with oligomeric and polymeric PA compared to amylopectin. The PA extract increased the net RS in normal starch by about 2 times more than the monomeric polyphenol extract; debranching amylopectin increased the difference by 4.3 times. Only treatments with PA increased RS in high amylose starch (52% higher than the control). Sorghum PAs interact strongly with starch, decreasing starch digestibility. The interactions appear to be specific to amylose and linear fragments of amylopectin, suggesting hydrophobic interactions are involved.

  6. Synthesis of α(1→4)-linked non-natural mannoglucans by α-glucan phosphorylase-catalyzed enzymatic copolymerization.

    PubMed

    Baba, Ryotaro; Yamamoto, Kazuya; Kadokawa, Jun-Ichi

    2016-10-20

    α-Glucan phosphorylase catalyzes enzymatic polymerization of α-d-glucose 1-phosphate (Glc-1-P) as a monomer from a maltooligosaccharide primer to produce α(1→4)-glucan, i.e., amylose, with liberating inorganic phosphate (Pi). Because of quite weak specificity for the recognition of substrates by thermostable α-glucan phosphorylase (from Aquifex aeolicus VF5), in this study, we investigated the enzymatic copolymerization of Glc-1-P with its analogue monomer, α-d-mannose 1-phosphate (Man-1-P) under the conditions for removal of Pi as the precipitate with ammonium and magnesium in ammonia buffer containing Mg(2+) ion to produce α(1→4)-linked non-natural mannoglucans composed of Glc/Man units. The reaction was conducted in different feed ratios using the maltotriose primer at 40°C for 7days. The MALDI-TOF mass and (1)H NMR spectra of the products fully supported the mannoglucan structures. PMID:27474652

  7. KIAA1199 interacts with glycogen phosphorylase kinase β-subunit (PHKB) to promote glycogen breakdown and cancer cell survival.

    PubMed

    Terashima, Masato; Fujita, Yoshihiko; Togashi, Yosuke; Sakai, Kazuko; De Velasco, Marco A; Tomida, Shuta; Nishio, Kazuto

    2014-08-30

    The KIAA1199 gene was first discovered to be associated with non-syndromic hearing loss. Recently, several reports have shown that the up-regulation of KIAA1199 is associated with cancer cell migration or invasion and a poor prognosis. These findings indicate that KIAA1199 may be a novel target for cancer therapy. Therefore, we explored in detail the function of KIAA1199 in cancer cells. In this study, we investigated the interaction of KIAA1199 protein with intracellular proteins in cancer cells. To this end, we expressed KIAA1199-MBP fusion protein and performed a pull-down assay. In addition, KIAA1199-overexpressing cancer cell lines were constructed using a retroviral vector and were used for further experiments. A pull-down analysis showed that the glycogen phosphorylase kinase β-subunit (PHKB) interacted with the C-terminal region of KIAA1199 protein. Furthermore, we observed the interaction of KIAA1199 with glycogen phosphorylase brain form (PYGB) under serum-free conditions. The interaction promoted glycogen breakdown and cancer cell survival. Our findings indicate that KIAA1199 plays an important role in glycogen breakdown and cancer cell survival and that it may represent a novel target for cancer therapy.

  8. Quantitative description of the absorption spectra of the coenzyme in glycogen phosphorylases based on log-normal distribution curves.

    PubMed Central

    Donoso, J; Muñoz, F; Garcia Blanco, F

    1993-01-01

    The absorption spectra of the coenzyme [pyridoxal 5'-phosphate (PLP)] in glycogen phosphorylase a (GPha), glycogen phosphorylase b (GPhb) and of the latter bound to various effectors and substrates were analysed on the basis of log-normal distribution curves. The results obtained showed that the ionization state of the PLP and GPha environment differs from that of GPhb. This divergence was interpreted in terms of tautomeric equilibria between some forms of the Schiff base of PLP and enzymic Lys-679. The ionic forms are slightly more predominant in GPha than they are in GPhb, so ionic and/or hydrogen-bonding interactions between the aromatic ring of PLP and GPha must be stronger than with GPhb. This confirms the purely structural role of the aromatic ring of the coenzyme. Binding of GPhb to AMP and Mg2+ results in the coenzyme adopting a similar state as in GPha. On the other hand, binding to IMP gives rise to no detectable changes in the tautomeric equilibrium of the coenzyme. PMID:8503849

  9. KIAA1199 interacts with glycogen phosphorylase kinase β-subunit (PHKB) to promote glycogen breakdown and cancer cell survival

    PubMed Central

    Terashima, Masato; Fujita, Yoshihiko; Togashi, Yosuke; Sakai, Kazuko; De Velasco, Marco A.; Tomida, Shuta; Nishio, Kazuto

    2014-01-01

    The KIAA1199 gene was first discovered to be associated with non-syndromic hearing loss. Recently, several reports have shown that the up-regulation of KIAA1199 is associated with cancer cell migration or invasion and a poor prognosis. These findings indicate that KIAA1199 may be a novel target for cancer therapy. Therefore, we explored in detail the function of KIAA1199 in cancer cells. In this study, we investigated the interaction of KIAA1199 protein with intracellular proteins in cancer cells. To this end, we expressed KIAA1199-MBP fusion protein and performed a pull-down assay. In addition, KIAA1199-overexpressing cancer cell lines were constructed using a retroviral vector and were used for further experiments. A pull-down analysis showed that the glycogen phosphorylase kinase β-subunit (PHKB) interacted with the C-terminal region of KIAA1199 protein. Furthermore, we observed the interaction of KIAA1199 with glycogen phosphorylase brain form (PYGB) under serum-free conditions. The interaction promoted glycogen breakdown and cancer cell survival. Our findings indicate that KIAA1199 plays an important role in glycogen breakdown and cancer cell survival and that it may represent a novel target for cancer therapy. PMID:25051373

  10. Glucose-derived spiro-isoxazolines are anti-hyperglycemic agents against type 2 diabetes through glycogen phosphorylase inhibition.

    PubMed

    Goyard, David; Kónya, Bálint; Chajistamatiou, Aikaterini S; Chrysina, Evangelia D; Leroy, Jérémy; Balzarin, Sophie; Tournier, Michel; Tousch, Didier; Petit, Pierre; Duret, Cédric; Maurel, Patrick; Somsák, László; Docsa, Tibor; Gergely, Pál; Praly, Jean-Pierre; Azay-Milhau, Jacqueline; Vidal, Sébastien

    2016-01-27

    Glycogen phosphorylase (GP) is a target for the treatment of hyperglycaemia in the context of type 2 diabetes. This enzyme is responsible for the depolymerization of glycogen into glucose thereby affecting the levels of glucose in the blood stream. Twelve new d-glucopyranosylidene-spiro-isoxazolines have been prepared from O-peracylated exo-D-glucals by regio- and stereoselective 1,3-dipolar cycloaddition of nitrile oxides generated in situ by treatment of the corresponding oximes with bleach. This mild and direct procedure appeared to be applicable to a broad range of substrates. The corresponding O-unprotected spiro-isoxazolines were evaluated as glycogen phosphorylase (GP) inhibitors and exhibited IC50 values ranging from 1 to 800 μM. Selected inhibitors were further evaluated in vitro using rat and human hepatocytes and exhibited significant inhibitory properties in the primary cell culture. Interestingly, when tested with human hepatocytes, the tetra-O-acetylated spiro-isoxazoline bearing a 2-naphthyl residue showed a much lower IC50 value (2.5 μM), compared to that of the O-unprotected analog (19.95 μM). The most promising compounds were investigated in Zucker fa/fa rat model in acute and sub-chronic assays and decreased hepatic glucose production, which is known to be elevated in type 2 diabetes. This indicates that glucose-based spiro-isoxazolines can be considered as anti-hyperglycemic agents in the context of type 2 diabetes.

  11. Plastidic phosphoglucomutase and ADP-glucose pyrophosphorylase mutants impair starch synthesis in rice pollen grains and cause male sterility

    PubMed Central

    Lee, Sang-Kyu; Eom, Joon-Seob; Hwang, Seon-Kap; Shin, Dongjin; An, Gynheung; Okita, Thomas W.; Jeon, Jong-Seong

    2016-01-01

    To elucidate the starch synthesis pathway and the role of this reserve in rice pollen, we characterized mutations in the plastidic phosphoglucomutase, OspPGM, and the plastidic large subunit of ADP-glucose (ADP-Glc) pyrophosphorylase, OsAGPL4. Both genes were up-regulated in maturing pollen, a stage when starch begins to accumulate. Progeny analysis of self-pollinated heterozygous lines carrying the OspPGM mutant alleles, osppgm-1 and osppgm-2, or the OsAGPL4 mutant allele, osagpl4-1, as well as reciprocal crosses between the wild type (WT) and heterozygotes revealed that loss of OspPGM or OsAGPL4 caused male sterility, with the former condition rescued by the introduction of the WT OspPGM gene. While iodine staining and transmission electron microscopy analyses of pollen grains from homozygous osppgm-1 lines produced by anther culture confirmed the starch null phenotype, pollen from homozygous osagpl4 mutant lines, osagpl4-2 and osagpl4-3, generated by the CRISPR/Cas system, accumulated small amounts of starch which were sufficient to produce viable seed. Such osagpl4 mutant pollen, however, was unable to compete against WT pollen successfully, validating the important role of this reserve in fertilization. Our results demonstrate that starch is mainly polymerized from ADP-Glc synthesized from plastidic hexose phosphates in rice pollen and that starch is an essential requirement for successful fertilization in rice. PMID:27588462

  12. Anthranilimide-based glycogen phosphorylase inhibitors for the treatment of type 2 diabetes: 1. Identification of 1-amino-1-cycloalkyl carboxylic acid headgroups

    SciTech Connect

    Sparks, Steven M.; Banker, Pierette; Bickett, David M.; Carter, H. Luke; Clancy, Daphne C.; Dickerson, Scott H.; Dwornik, Kate A.; Garrido, Dulce M.; Golden, Pamela L.; Nolte, Robert T.; Peat, Andrew J.; Sheckler, Lauren R.; Tavares, Francis X.; Thomson, Stephen A.; Wang, Liping; Weiel, James E.

    2009-05-15

    Optimization of the amino acid residue within a series of anthranilimide-based glycogen phosphorylase inhibitors is described. These studies culminated in the identification of anthranilimides 16 and 22 which displayed potent in vitro inhibition of GPa in addition to reduced inhibition of CYP2C9 and excellent pharmacokinetic properties.

  13. Expression of a cDNA for the catalytic subunit of skeletal-muscle phosphorylase kinase in transfected 3T3 cells.

    PubMed Central

    Cawley, K C; Akita, C G; Walsh, D A

    1989-01-01

    Phosphorylase kinase is a multimeric enzyme of composition (alpha, beta, gamma, delta)4 whose catalytic activity resides in the gamma-subunit. As an approach to understand further its regulation, a cDNA for the gamma-subunit of phosphorylase kinase (gamma PhK) has been cloned into a mammalian expression vector behind the mouse metallothionein-1 promoter. NIH 3T3 cells were co-transfected with this construct (pEV gamma PhK) and pSV2neo, G418-resistant clones were selected, and several were found to have stably incorporated the gamma-subunit cDNA into their genomic DNA. Phosphorylase kinase activity was clearly present in extracts from cultures of pEV gamma PhK-transformed cells and increased several-fold after 24 h of incubation with Zn2+, whereas it was undetectable in the parent 3T3 cells. A significant, but variable, proportion (15-70%) of the activity was Ca2+-dependent. We conclude that the phosphorylase kinase activity expressed by the cells transformed with pEV gamma PhK is due to free gamma-subunit and gamma-subunit associated with cellular calmodulin, which replaces the delta-subunit normally associated with the gamma-subunit in the holoenzyme. Images Fig. 1. Fig. 2. Fig. 3. PMID:2481439

  14. Molecular disassembly of rice and lotus starches during thermal processing and its effect on starch digestibility.

    PubMed

    Wang, Shujun; Sun, Yue; Wang, Jinrong; Wang, Shuo; Copeland, Les

    2016-02-01

    The molecular disassembly of starch during thermal processing is a major determinant for the susceptibility of starch to enzymatic digestion. In the present study, the effects of thermal processing on the disassembly of the granular structure and the in vitro enzymatic digestibility of rice and lotus starches were investigated. After heating at 50 °C, rice and lotus starches did not show significant changes in granular morphology, long-range crystallinity and short-range molecular order. As the temperature increased to 60 °C, rice starch underwent a partial gelatinization followed by an incomplete disruption of granular morphology, crystallites and molecular order. In contrast, lotus starch was almost completely gelatinized at 60 °C. At 70 °C or higher, both starches were fully gelatinized with complete disruption of the micro and macro structures. Our results show that gelatinization greatly increased the in vitro enzymatic digestibility of both starches, but that the degree of disassembly of the starch structure during thermal processing was not a major determinant of the digestibility of gelatinized starch. PMID:26829664

  15. Starch Origin and Thermal Processing Affect Starch Digestion in a Minipig Model of Pancreatic Exocrine Insufficiency

    PubMed Central

    Mößeler, Anne; Vagt, Sandra; Beyerbach, Martin; Kamphues, Josef

    2015-01-01

    Although steatorrhea is the most obvious symptom of pancreatic exocrine insufficiency (PEI), enzymatic digestion of protein and starch is also impaired. Low praecaecal digestibility of starch causes a forced microbial fermentation accounting for energy losses and meteorism. To optimise dietetic measures, knowledge of praecaecal digestibility of starch is needed but such information from PEI patients is rare. Minipigs fitted with an ileocaecal fistula with (n = 3) or without (n = 3) pancreatic duct ligation (PL) were used to estimate the rate of praecaecal disappearance (pcD) of starch. Different botanical sources of starch (rice, amaranth, potato, and pea) were fed either raw or cooked. In the controls (C), there was an almost complete pcD (>92%) except for potato starch (61.5%) which was significantly lower. In PL pcD of raw starch was significantly lower for all sources of starch except for amaranth (87.9%). Thermal processing increased pcD in PL, reaching values of C for starch from rice, potato, and pea. This study clearly underlines the need for precise specification of starch used for patients with specific dietetic needs like PEI. Data should be generated in suitable animal models or patients as tests in healthy individuals would not have given similar conclusions. PMID:26064101

  16. Molecular disassembly of rice and lotus starches during thermal processing and its effect on starch digestibility.

    PubMed

    Wang, Shujun; Sun, Yue; Wang, Jinrong; Wang, Shuo; Copeland, Les

    2016-02-01

    The molecular disassembly of starch during thermal processing is a major determinant for the susceptibility of starch to enzymatic digestion. In the present study, the effects of thermal processing on the disassembly of the granular structure and the in vitro enzymatic digestibility of rice and lotus starches were investigated. After heating at 50 °C, rice and lotus starches did not show significant changes in granular morphology, long-range crystallinity and short-range molecular order. As the temperature increased to 60 °C, rice starch underwent a partial gelatinization followed by an incomplete disruption of granular morphology, crystallites and molecular order. In contrast, lotus starch was almost completely gelatinized at 60 °C. At 70 °C or higher, both starches were fully gelatinized with complete disruption of the micro and macro structures. Our results show that gelatinization greatly increased the in vitro enzymatic digestibility of both starches, but that the degree of disassembly of the starch structure during thermal processing was not a major determinant of the digestibility of gelatinized starch.

  17. 21 CFR 178.3520 - Industrial starch-modified.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Industrial starch-modified. 178.3520 Section 178... SANITIZERS Certain Adjuvants and Production Aids § 178.3520 Industrial starch-modified. Industrial starch... provisions of this section. (a) Industrial starch-modified is identified as follows: (1) A food...

  18. 21 CFR 178.3520 - Industrial starch-modified.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Industrial starch-modified. 178.3520 Section 178... SANITIZERS Certain Adjuvants and Production Aids § 178.3520 Industrial starch-modified. Industrial starch... provisions of this section. (a) Industrial starch-modified is identified as follows: (1) A food...

  19. 21 CFR 178.3520 - Industrial starch-modified.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Industrial starch-modified. 178.3520 Section 178... § 178.3520 Industrial starch-modified. Industrial starch-modified may be safely used as a component of..., transporting, or holding food, subject to the provisions of this section. (a) Industrial starch-modified...

  20. Structure and Mechanism of an ADP-Glucose Phosphorylase from Arabidopsis thaliana†,‡

    PubMed Central

    McCoy, Jason G.; Arabshahi, Abolfazl; Bitto, Eduard; Bingman, Craig A.; Ruzicka, Frank J.; Frey, Perry A.; Phillips, George N.

    2008-01-01

    The X-ray crystal structure of the At5g18200.1 protein has been solved to a nominal resolution of 2.30 Å. The structure has a histidine triad (HIT)-like fold containing two distinct HIT-like motifs. The sequence of At5g18200.1 indicates a distant family relationship to the Escherichia coli galactose-1-P uridylyltransferase (GalT): the determined structure of the At5g18200.1 protein confirms this relationship. The At5g18200.1 protein does not demonstrate GalT activity but instead catalyzes adenylyltransfer in the reaction of ADP-glucose with various phosphates. The best acceptor among those evaluated is phosphate itself, thus the At5g18200.1 enzyme appears to be an ADP-glucose phosphorylase. The enzyme catalyzes the exchange of 14C between ADP-[14C]glucose and glucose-1-P in the absence of phosphate. The steady state kinetics of exchange follows the ping pong bi bi kinetic mechanism, with kcat = 4.1 s−1 and Km-values of 1.4 µM and 83 µM for ADP-[14C]glucose and glucose-1-P, respectively, at pH 8.5 and 25 °C. The overall reaction of ADP-glucose with phosphate to produce ADP and glucose-1-P follows ping pong bi bi steady state kinetics, with kcat = 2.7 s−1 and Km-values of 6.9 µM and 90 µM for ADP-glucose and phosphate, respectively, at pH 8.5 and 25 °C. The kinetics are consistent with a double displacement mechanism that involves a covalent adenylyl-enzyme intermediate. The X-ray crystal structure of this intermediate was solved to 1.83 Å resolution, and shows the AMP-group bonded to His186. The value of Keq in the direction of ADP and glucose-1-P formation is 5.0 at pH 7.0 and 25 °C in the absence of a divalent metal ion, and it is 40 in the presence of 1 mM MgCl2. PMID:16519510

  1. Model approach to starch functionality in bread making.

    PubMed

    Goesaert, Hans; Leman, Pedro; Delcour, Jan A

    2008-08-13

    We used modified wheat starches in gluten-starch flour models to study the role of starch in bread making. Incorporation of hydroxypropylated starch in the recipe reduced loaf volume and initial crumb firmness and increased crumb gas cell size. Firming rate and firmness after storage increased for loaves containing the least hydroxypropylated starch. Inclusion of cross-linked starch had little effect on loaf volume or crumb structure but increased crumb firmness. The firming rate was mostly similar to that of control samples. Presumably, the moment and extent of starch gelatinization and the concomitant water migration influence the structure formation during baking. Initial bread firmness seems determined by the rigidity of the gelatinized granules and leached amylose. Amylopectin retrogradation and strengthening of a long-range network by intensifying the inter- and intramolecular starch-starch and possibly also starch-gluten interactions (presumably because of water incorporation in retrograded amylopectin crystallites) play an important role in firming.

  2. Native starch in tablet formulations: properties on compaction.

    PubMed

    Bos, C E; Bolhuis, G K; Van Doorne, H; Lerk, C F

    1987-10-16

    Maize, potato, rice and tapioca (cassava) starch were evaluated with respect to their properties on direct compression. Rice starch showed much better compactibility as compared to maize, potato and tapioca starch. Moreover, its binding capacity proved to be almost insensitive to mixing with magnesium stearate. This in contrast to the dramatic decrease in crushing strength of potato starch tablets containing the lubricant. The compactibility of the starches was found to be strongly affected by the equilibrium moisture content of the starches, which is dependent on the relative humidity of the atmosphere under which the powders were stored. All starches showed adequate capacity for water uptake to act as a disintegrant. Rice starch exhibited worst flowability, caused by its fine particle size as compared to the other starches. Granulation of rice starch changed it into a potential filler-binder in tablets prepared by direct compression.

  3. Plant-crafted starches for bioplastics production.

    PubMed

    Sagnelli, Domenico; Hebelstrup, Kim H; Leroy, Eric; Rolland-Sabaté, Agnès; Guilois, Sophie; Kirkensgaard, Jacob J K; Mortensen, Kell; Lourdin, Denis; Blennow, Andreas

    2016-11-01

    Transgenically-produced amylose-only (AO) starch was used to manufacture bioplastic prototypes. Extruded starch samples were tested for crystal residues, elasticity, glass transition temperature, mechanical properties, molecular mass and microstructure. The AO starch granule crystallinity was both of the B- and Vh-type, while the isogenic control starch was mainly A-type. The first of three endothermic transitions was attributed to gelatinization at about 60°C. The second and third peaks were identified as melting of the starch and amylose-lipid complexes, respectively. After extrusion, the AO samples displayed Vh- and B-type crystalline structures, the B-type polymorph being the dominant one. The AO prototypes demonstrated a 6-fold higher mechanical stress at break and 2.5-fold higher strain at break compared to control starch. Dynamic mechanical analysis showed a significant increase in the storage modulus (E') for AO samples compared to the control. The data support the use of pure starch-based bioplastics devoid of non-polysaccharide fillers.

  4. Plant-crafted starches for bioplastics production.

    PubMed

    Sagnelli, Domenico; Hebelstrup, Kim H; Leroy, Eric; Rolland-Sabaté, Agnès; Guilois, Sophie; Kirkensgaard, Jacob J K; Mortensen, Kell; Lourdin, Denis; Blennow, Andreas

    2016-11-01

    Transgenically-produced amylose-only (AO) starch was used to manufacture bioplastic prototypes. Extruded starch samples were tested for crystal residues, elasticity, glass transition temperature, mechanical properties, molecular mass and microstructure. The AO starch granule crystallinity was both of the B- and Vh-type, while the isogenic control starch was mainly A-type. The first of three endothermic transitions was attributed to gelatinization at about 60°C. The second and third peaks were identified as melting of the starch and amylose-lipid complexes, respectively. After extrusion, the AO samples displayed Vh- and B-type crystalline structures, the B-type polymorph being the dominant one. The AO prototypes demonstrated a 6-fold higher mechanical stress at break and 2.5-fold higher strain at break compared to control starch. Dynamic mechanical analysis showed a significant increase in the storage modulus (E') for AO samples compared to the control. The data support the use of pure starch-based bioplastics devoid of non-polysaccharide fillers. PMID:27516287

  5. 75 FR 879 - National Starch and Chemical Company Specialty Starches Division Including On-Site Leased Workers...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-01-06

    ..., applicable to workers of National Starch and Chemical Company, Specialty Starches Division, Island Falls, Maine. The notice was published in the Federal Register on December 31, 2007 (72 FR 74343). At the... Employment and Training Administration National Starch and Chemical Company Specialty Starches...

  6. Effects of salts on the gelatinization and retrogradation properties of maize starch and waxy maize starch.

    PubMed

    Wang, Wei; Zhou, Hongxian; Yang, Hong; Zhao, Siming; Liu, Youming; Liu, Ru

    2017-01-01

    The objective of this study was to evaluate the effects of salts on the gelatinization and retrogradation of maize and waxy maize starch. Experimental results showed that the salting-out or structure-making ions, such as F(-) and SO4(2-), decreased the swelling power, solubility and transparency of both starches, but increased the gelatinization temperature, enthalpy, and syneresis, due to the tendency of these ions to protect the hydrogen bond links among starch molecules. On the other hand, the salting-in or structure-breaking ions, such as I(-) and SCN(-), exhibited the opposite effects. Microscopic observations confirmed such effects of salts on both starches. Furthermore, the effects of salts were more significant on waxy maize and on normal maize starch. Generally, salts could significantly influence on the gelatinization and retrogradation of maize and waxy maize starch, following the order of the Hofmeister series. PMID:27507481

  7. Applicability, Commercial Utility and Recent Patents on Starch and Starch Derivative as Pharmaceutical Drug Delivery Carrier.

    PubMed

    Pandey, Shreya; Malviya, Rishabha; Sharma, Pramod K

    2015-01-01

    Natural polymers are widely utilized in pharmaceutical and food industries. Starch, a major carbohydrate is a staple food in human and animal diets which is simply extractable from various sources, like potato, maize, corn, wheat, etc. It is widely used as a raw material in various food and non food industries as well as in paper, textile and other industries. This article summarizes the starch and modification of starch and to produce a novel molecule with various applications in industries including number of advances in pharmaceutical industry. The unique characteristics of starch and their modified form can be successfully used as drug delivery carriers in various pharmaceutical preparations. It is widely used as controlled and sustained release polymer, tablet disintegrant, drug delivery carrier, plasma volume expander and also finds its applicability in bone tissue engineering and in artificial red cells. It also includes the patents related to starch and modified starch based products and their commercial utility. PMID:26205680

  8. Starch removal from potato tuber sections.

    PubMed

    Fronda, A; Jona, R

    1991-01-01

    Heating plant sections at 90 C with 0.5% aqueous ammonium oxalate is required to remove pectins. When applied to tissues rich in starch such as potato, this step produces heavy dextrinization of the starch which hinders subsequent evaluation of the extinction values of the cell walls. To overcome this a method has been devised to brush away the starch granules from the sections with a thin paint brush, just after paraffin removal by xylene. The slide is then processed as usual: pectins are removed by heat treatment, cell walls are stained with PAS and the stain intensity can be evaluated by photometry. PMID:1790235

  9. Ion mobility-mass spectrometry of phosphorylase B ions generated with supercharging reagents but in charge-reducing buffer.

    PubMed

    Hogan, Christopher J; Ogorzalek Loo, Rachel R; Loo, Joseph A; de la Mora, Juan Fernandez

    2010-11-01

    We investigate whether "supercharging" reagents able to shift the charge state distributions (CSDs) of electrosprayed protein ions upward also influence gas-phase protein structure. A differential mobility analyzer and a mass spectrometer are combined in series (DMA-MS) to measure the mass and mobility of monomer and multimeric phosphorylase B ions (monomer molecular weight ∼97 kDa) in atmospheric pressure air. Proteins are electrosprayed from charge-reducing triethylammonium formate in water (pH = 6.8) with and without the addition of the supercharging reagent tetramethylene sulfone (sulfolane). Because the DMA measures ion mobility prior to collisional heating or declustering, it probes the structure of supercharged protein ions immediately following solvent (water) evaporation. As in prior studies, the addition of sulfolane is found to drastically increase both the mean and maximum charge state of phosphorylase B ions. Ions from all protein n-mers were found to yield mobilities that, for a given charge state, were ∼6-10% higher in the absence of sulfolane. We find that the mobility decrease which arises with sulfolane is substantially smaller than that typically observed for folded-to-unfolded transitions in protein ions (where a ∼60% decrease in mobility is typical), suggesting that supercharging reagents do not cause structural protein modifications in solution as large as noted recently by Williams and colleagues [E. R. Williams et al., J. Am. Soc. Mass Spectrom., 2010, 21, 1762-1774]. In fact, the measurements described here indicate that the modest mobility decrease observed can be partly attributed to sulfolane trapping within the protein ions during DMA measurements, and probably also in solution. As the most abundant peaks in measured mass-mobility spectra for ions produced with and without sulfolane correspond to non-covalently bound phosphorylase B dimers, we find that in spite of a change in mobility/cross section, sulfolane addition does not

  10. Resistant starch improvement of rice starches under a combination of acid and heat-moisture treatments.

    PubMed

    Hung, Pham Van; Vien, Ngo Lam; Lan Phi, Nguyen Thi

    2016-01-15

    The effects of a combination of acid and heat-moisture treatment on formation of resistant starch (RS) and characteristics of high-amylose, normal and waxy rice starches were investigated in this study. The degrees of polymerization of the rice starches treated with citric acid, lactic acid or acetic acid were significantly reduced as compared to the native starches. The RS contents of acid and heat-moisture treated rice starches were in a range of 30.1-39.0%, significantly higher than those of native rice starches (6.3-10.2%) and those of heat-moisture treated rice starches (18.5-23.9%). The acid and heat-moisture treatments reduced swelling power and viscosity, but increased solubility of the starches, while the crystalline structure did not change. Among the organic acids used, citric acid had the most impact on starch characteristics and RS formation, followed by lactic acid and acetic acid. The results are useful in production of RS for functional food application. PMID:26258703

  11. Resistant starch improvement of rice starches under a combination of acid and heat-moisture treatments.

    PubMed

    Hung, Pham Van; Vien, Ngo Lam; Lan Phi, Nguyen Thi

    2016-01-15

    The effects of a combination of acid and heat-moisture treatment on formation of resistant starch (RS) and characteristics of high-amylose, normal and waxy rice starches were investigated in this study. The degrees of polymerization of the rice starches treated with citric acid, lactic acid or acetic acid were significantly reduced as compared to the native starches. The RS contents of acid and heat-moisture treated rice starches were in a range of 30.1-39.0%, significantly higher than those of native rice starches (6.3-10.2%) and those of heat-moisture treated rice starches (18.5-23.9%). The acid and heat-moisture treatments reduced swelling power and viscosity, but increased solubility of the starches, while the crystalline structure did not change. Among the organic acids used, citric acid had the most impact on starch characteristics and RS formation, followed by lactic acid and acetic acid. The results are useful in production of RS for functional food application.

  12. Resistant-starch formation in high-amylose maize starch during Kernel development.

    PubMed

    Jiang, Hongxin; Lio, Junyi; Blanco, Mike; Campbell, Mark; Jane, Jay-Lin

    2010-07-14

    The objective of this study was to understand the resistant-starch (RS) formation during kernel development of a high-amylose maize, GEMS-0067 line. The RS content of the starch, determined using AOAC method 991.43 for total dietary fiber, increased with kernel maturation and increase in the amylose/intermediate component (IC) content of the starch. Gelatinization of the native starches showed a major thermal transition with peak temperature at 76.6-81.0 degrees C. An additional peak ( approximately 97.1 degrees C) first appeared 20 days after pollination and then developed into a significant peak on later dates. After removal of lipids from the starch, this peak disappeared, but the conclusion gelatinization temperature remained the same. The proportion of the enthalpy change of the thermal transition above 95 degrees C, calculated from the thermogram of the defatted starch, increased with kernel maturation and was significantly correlated with the RS content of the starch (r = 0.98). These results showed that the increase in crystallites of amylose/IC long-chain double helices in the starch resulted in the increase in the RS content of the starch during kernel development.

  13. Dilute solution properties of canary seed (Phalaris canariensis) starch in comparison to wheat starch.

    PubMed

    Irani, Mahdi; Razavi, Seyed M A; Abdel-Aal, El-Sayed M; Hucl, Pierre; Patterson, Carol Ann

    2016-06-01

    Dilute solution properties of an unknown starch are important to understand its performance and applications in food and non-food industries. In this paper, rheological and molecular properties (intrinsic viscosity, molecular weight, shape factor, voluminosity, conformation and coil overlap parameters) of the starches from two hairless canary seed varieties (CO5041 & CDC Maria) developed for food use were evaluated in the dilute regime (Starch dispersions in DMSO (0.5g/dl)) and compared with wheat starch (WS). The results showed that Higiro model is the best among five applied models for intrinsic viscosity determination of canary seed starch (CSS) and WS on the basis of coefficient of determination (R(2)) and root mean square error (RMSE). WS sample showed higher intrinsic viscosity value (1.670dl/g) in comparison to CSS samples (1.325-1.397dl/g). Berry number and the slope of master curve demonstrated that CSS and WS samples were in dilute domain without entanglement occurrence. The shape factor suggested spherical and ellipsoidal structure for CO5041 starch and ellipsoidal for CDC Maria starch and WS. The molecular weight, coil radius and coil volume of CSSs were smaller than WS. The behavior and molecular characterization of canary seed starch showed its unique properties compared with wheat starch.

  14. Electron microscopy and composition of raw acorn starch in relation to in vivo starch digestibility.

    PubMed

    Cappai, Maria Grazia; Alesso, Giuseppe Andrea; Nieddu, Giuseppa; Sanna, Marina; Pinna, Walter

    2013-06-01

    The structure and composition of starch play an important role as co-factors affecting raw starch digestibility: such features were investigated in raw acorn starch from the most diffused oak trees in the Mediterranean basin. A total of 620 whole ripe acorns from Holm (Quercus ilex L., n = 198), Downy (Quercus pubescens Willd., n = 207) and Cork (Quercus suber L., n = 215) oaks sampled on the Sardinia Isle (40° 56' 0'' N; 9° 4' 0'' E; 545 m above the mean sea level) in the same geographical area, were analyzed for their chemical composition. The starch contents ranged between 51.2% and 53.5% of dry matter. The starch granules displayed a spheroid/ovoid and cylindrical shape; on scanning electron microscopic (SEM) analyses, a bimodal distribution of starch granule size was observed both for Holm and Cork oak acorns, whereas the starch granules of Downy oak acorns showed diameters between 10.2 and 13.8 μm. The specific amylose to amylopectin ratio of acorn starch was 25.8%, 19.5% and 34.0% in the Holm, Downy and Cork oaks, respectively. The (13)C Nuclear Magnetic Resonance (NMR) signal analysis displayed a pivotal spectrum for the identification of the amylose peaks in raw acorn starch, as a basis for the amylose to amylopectin ratio determination.

  15. Cooking behavior and starch digestibility of NUTRIOSE® (resistant starch) enriched noodles from sweet potato flour and starch.

    PubMed

    Menon, Renjusha; Padmaja, G; Sajeev, M S

    2015-09-01

    The effect of a resistant starch source, NUTRIOSE® FB06 at 10%, 15% and 20% in sweet potato flour (SPF) and 5% and 10% in sweet potato starch (SPS) in reducing the starch digestibility and glycaemic index of noodles was investigated. While NUTRIOSE (10%) significantly reduced the cooking loss in SPF noodles, this was enhanced in SPS noodles and guar gum (GG) supplementation reduced CL of both noodles. In vitro starch digestibility (IVSD) was significantly reduced in test noodles compared to 73.6g glucose/100g starch in control SPF and 65.9 g in SPS noodles. Resistant starch (RS) was 54.96% for NUTRIOSE (15%)+GG (1%) fortified SPF noodles and 53.3% for NUTRIOSE (5%)+GG (0.5%) fortified SPS noodles, as against 33.8% and 40.68%, respectively in SPF and SPS controls. Lowest glycaemic index (54.58) and the highest sensory scores (4.23) were obtained for noodles with 15% NUTRIOSE+1% GG.

  16. Physicochemical and binder properties of starch obtained from Cyperus esculentus.

    PubMed

    Manek, Rahul V; Builders, Philip F; Kolling, William M; Emeje, Martins; Kunle, Olobayo O

    2012-06-01

    The purpose of this study was to isolate starch from the tubers of Cyperus esculentus L. and evaluate its physicochemical and binder properties. Extraction of starch using sodium metabisulfite yielded 37 g of starch per 100 g of the tubers. Scanning electron microscopy indicated that Cyperus starch consists of oval to elliptical particles with a smooth surface. Cyperus starch demonstrates a narrow particle size distribution with a mean of 8.25 μm. Cyperus starch conforms well to United States Pharmacopeia standards established for widely used starches like maize and potato. The X-ray powder diffraction pattern and moisture sorption profile of Cyperus starch were comparable to that of maize starch. Cyperus starch had lower swelling power than maize and potato starch, indicative of stronger associative forces within the granules. Carr's index and Hausner ratio indicate that Cyperus starch should have comparable flow properties with respect to maize and potato starch. Cyperus starch was employed as binder for the formulation of metronidazole tablets. Formulations containing 5%, 7.5%, and 10% Cyperus starch were compared with those containing 10% potato starch. At 10% binder concentration, the tablets containing Cyperus starch exhibited better hardness and negligible friability as compared with those with potato starch. Although the binder concentration had a significant effect on the disintegration time of the tablets, it did not seem to affect the dissolution profile. These results indicate that Cyperus starch provides excellent binding properties without compromising drug release characteristics and should be explored in pharmaceutical formulations. PMID:22350737

  17. Functional characterization of three (GH13) branching enzymes involved in cyanobacterial starch biosynthesis from Cyanobacterium sp. NBRC 102756.

    PubMed

    Suzuki, Ryuichiro; Koide, Keiichi; Hayashi, Mari; Suzuki, Tomoko; Sawada, Takayuki; Ohdan, Takashi; Takahashi, Hidekazu; Nakamura, Yasunori; Fujita, Naoko; Suzuki, Eiji

    2015-05-01

    Starch and glycogen are widespread storage polysaccharides in bacteria, plants, and animals. Recently, some cyanobacteria were found to accumulate water-insoluble α-glucan similar to amylopectin rather than glycogen, the latter of which is more commonly produced in these organisms. The amylopectin-producing species including Cyanobacterium sp. NBRC 102756 invariably have three branching enzyme (BE) homologs, BE1, BE2, and BE3, all belonging to the glycoside hydrolase family 13. Multiple BE isoforms in prokaryotes have not been previously studied. In the present work, we carried out functional characterization of these enzymes expressed in Escherichia coli. The recombinant enzymes were all active, although the specific activity of BE3 was much lower than those of BE1 and BE2. After the incubation of the enzymes with amylopectin or amylose, the reaction products were analyzed by fluorophore-assisted carbohydrate capillary electrophoresis method. BE1 and BE2 showed similar chain-length preference to BEIIb isoform of rice (Oryza sativa L.), while the catalytic specificity of BE3 was similar to that of rice BEI. These results indicate that starch-producing cyanobacteria have both type-I BE (BE3) and type-II BEs (BE1 and BE2) in terms of chain-length preferences, as is the case of plants. All BE isoforms were active against phosphorylase limit dextrin, in which outer branches had been uniformly diminished to 4 glucose residues. Based on its catalytic properties, BE3 was assumed to have a role to transfer the glucan chain bearing branch(es) to give rise to a newly growing unit, or cluster as observed in amylopectin molecule.

  18. Biotechnological relevance of starch-degrading enzymes

    SciTech Connect

    Stewart, G.G.

    1987-01-01

    Traditional enzymes, such as the amylases and the proteases, have been improved, novel applications have been found and new and valuable products have been marketed. The enzymatic hydrolysis of starch is described in some detail. (Refs. 8).

  19. Production of modified starches by gamma irradiation

    NASA Astrophysics Data System (ADS)

    Kang, Il-Jun; Byun, Myung-Woo; Yook, Hong-Sun; Bae, Chun-Ho; Lee, Hyun-Soo; Kwon, Joong-Ho; Chung, Cha-Kwon

    1999-04-01

    As a new processing method for the production of modified starch, gamma irradiation and four kinds of inorganic peroxides were applied to commercial corn starch. The addition of inorganic peroxides without gamma irradiation or gamma irradiation without the addition of inorganic peroxides effectively decreased initial viscosity, but did not sufficiently keep viscosity stable. The combination of adding ammonium persulfate (APS) and gamma irradiation showed the lowest initial viscosity and the best stability out of the tested four kinds of inorganic peroxides. Among the tested mixing methods of APS, soaking was found to be more effective than dry blending or spraying. Therefore, the production of modified starch with low viscosity as well as with sufficient viscosity stability became feasible by the control of gamma irradiation dose levels and the amount of added APS to starch.

  20. Paint removal using wheat starch blast media

    NASA Astrophysics Data System (ADS)

    Foster, Terry; Oestreich, John

    1993-03-01

    A review of the Wheat Starch Blasting technology is presented. Laboratory evaluations covering Almen Arc testing on bare 2024-T3 aluminum and magnesium, as well as crack detection on 7075-T6 bare aluminum, are discussed. Comparisons with Type V plastic media show lower residual stresses are achieved on aluminum and magnesium with wheat starch media. Dry blasting effects on the detection of cracks confirms better crack visibility with wheat starch media versus Type V or Type II plastic media. Testing of wheat starch media in several composite test programs, including fiberglass, Kevlar, and graphite-epoxy composites, showed no fiber damage. Process developments and production experience at the first U.S. aircraft stripping facility are also reviewed. Corporate and regional aircraft are being stripped in this three nozzle dry blast hanger.

  1. Biodegradable starch-based polymeric materials

    NASA Astrophysics Data System (ADS)

    Suvorova, Anna I.; Tyukova, Irina S.; Trufanova, Elena I.

    2000-05-01

    The effects of low-molecular-weight additives, temperature and mechanical action on the structure and properties of starch are discussed. Special attention is given to mixtures of starch with synthetic polymers, e.g., co-polymers of ethylene with vinyl acetate, vinyl alcohol, acrylic acid, cellulose derivatives and other natural polymers. These mixtures can be used in the development of novel environmentally safe materials (films, coatings, packaging materials) and various articles for short-term use. The bibliography includes 105 references.

  2. Direct potentiometric determination of starch using a platinum redox sensor.

    PubMed

    Sakač, Nikola; Sak-Bosnar, Milan; Horvat, Marija

    2013-05-01

    Here, we describe the development of a platinum redox sensor for the direct potentiometric quantification of starch in solution. The sensor measures the decrease in free triiodide ion after it complexes with starch to form a starch-triiodide complex. This decrease was, therefore, correlated with starch concentration, and the composition and stability of the potassium triiodide solution were optimised. The starch-triiodide complex was characterized potentiometrically at variable starch and triiodide concentrations. We also propose a response mechanism for the platinum redox sensor towards starch and an appropriate theoretical model. The optimised method exhibited satisfactory accuracy and precision and was in good agreement with a standard spectrophotometric method. The sensor was tested over a range of 0.4-9 mg starch, with recoveries ranging from 97.8% to 103.4% and a detection limit of 0.01 mg starch.

  3. Radiation grafting of styrene on starch with high efficiency

    NASA Astrophysics Data System (ADS)

    Sheikh, N.; Akhavan, A.; Ataeivarjovi, E.

    2013-04-01

    Wheat starch grafted with polystyrene (PS-g-starch) was synthesized via polymerization grafting of styrene on starch by gamma-ray. The effects of starch/styrene weight ratio, and amount of applied doses (5-40 kGy) on the percentage of grafting, G (%), were investigated. The results showed that G (%) increased with increasing starch content. The optimum condition, starch/styrene weight ratio 1/3 and the applied dose 10 kGy, led to 252.9% of grafting. The obtained graft copolymer was characterized by Fourier transform infrared spectroscopy, X-ray diffraction, thermal gravimetric analysis, and scanning electron microscopy. FTIR spectroscopy as well as the XRD analysis exhibited the changes in chemical and crystalline structure of starch after grafting reaction. TGA demonstrated the changes in thermal stability of PS-g-starch copolymer. SEM micrographs indicated porous patches of PS adhering on the starch.

  4. Physicochemical properties and digestibility of hydrothermally treated waxy rice starch.

    PubMed

    Zeng, Feng; Ma, Fei; Kong, Fansheng; Gao, Qunyu; Yu, Shujuan

    2015-04-01

    Waxy rice starch was subjected to annealing (ANN) and heat-moisture treatment (HMT). These starches were also treated by a combination of ANN and HMT. The impact of single and dual modifications (ANN-HMT and HMT-ANN) on the molecular weight (M(w)), crystalline structure, thermal properties, and the digestibility were investigated. The relative crystallinity and short-range order on the granule surface increased on ANN, whereas decreased on HMT. All treated starches showed lower M(w) than that of the native starch. Gelatinization onset temperature, peak temperature and conclusion temperature increased for both single and dual treatments. Increased slowly digestible starch content was found on HMT and ANN-HMT. However, resistant starch levels decreased in all treated starches as compared with native starch. The results would imply that hydrothermal treatment induced structural changes in waxy rice starch significantly affected its digestibility.

  5. Heat expanded starch-based compositions.

    PubMed

    Glenn, Gregory M; Klamczynski, Artur K; Holtman, Kevin M; Shey, Justin; Chiou, Bor-Sen; Berrios, Jose; Wood, Delilah; Orts, William J; Imam, Syed H

    2007-05-16

    A heat expansion process similar to that used for expanded bead polystyrene was used to expand starch-based compositions. Foam beads made by solvent extraction had the appearance of polystyrene beads but did not expand when heated due to an open-cell structure. Nonporous beads, pellets, or particles were made by extrusion or by drying and milling cooked starch slurries. The samples expanded into a low-density foam by heating 190-210 degrees C for more than 20 s at ambient pressures. Formulations containing starch (50-85%), sorbitol (5-15%), glycerol (4-12%), ethylene vinyl alcohol (EVAL, 5-15%), and water (10-20%) were studied. The bulk density was negatively correlated to sorbitol, glycerol, and water content. Increasing the EVAL content increased the bulk density, especially at concentrations higher than 15%. Poly(vinyl alcohol) (PVAL) increased the bulk density more than EVAL. The bulk density was lowest in samples made of wheat and potato starch as compared to corn starch. The expansion temperature for the starch pellets decreased more than 20 degrees C as the moisture content was increased from 10 to 25%. The addition of EVAL in the formulations decreased the equilibrium moisture content of the foam and reduced the water absorption during a 1 h soaking period.

  6. The binding of β-d-glucopyranosyl-thiosemicarbazone derivatives to glycogen phosphorylase: A new class of inhibitors.

    PubMed

    Alexacou, Kyra-Melinda; Tenchiu Deleanu, Alia-Cristina; Chrysina, Evangelia D; Charavgi, Maria-Despoina; Kostas, Ioannis D; Zographos, Spyros E; Oikonomakos, Nikos G; Leonidas, Demetres D

    2010-11-15

    Glycogen phosphorylase (GP) is a promising target for the treatment of type 2 diabetes. In the process of structure based drug design for GP, a group of 15 aromatic aldehyde 4-(β-d-glucopyranosyl)thiosemicarbazones have been synthesized and evaluated as inhibitors of rabbit muscle glycogen phosphorylase b (GPb) by kinetic studies. These compounds are competitive inhibitors of GPb with respect to α-d-glucose-1-phosphate with IC(50) values ranging from 5.7 to 524.3μM. In order to elucidate the structural basis of their inhibition, the crystal structures of these compounds in complex with GPb at 1.95-2.23Å resolution were determined. The complex structures reveal that the inhibitors are accommodated at the catalytic site with the glucopyranosyl moiety at approximately the same position as α-d-glucose and stabilize the T conformation of the 280s loop. The thiosemicarbazone part of the studied glucosyl thiosemicarbazones possess a moiety derived from substituted benzaldehydes with NO(2), F, Cl, Br, OH, OMe, CF(3), or Me at the ortho-, meta- or para-position of the aromatic ring as well as a moiety derived from 4-pyridinecarboxaldehyde. These fit tightly into the β-pocket, a side channel from the catalytic site with no access to the bulk solvent. The differences in their inhibitory potency can be interpreted in terms of variations in the interactions of the aldehyde-derived moiety with protein residues in the β-pocket. In addition, 14 out of the 15 studied inhibitors were found bound at the new allosteric site of the enzyme.

  7. Thymidine phosphorylase in cancer cells stimulates human endothelial cell migration and invasion by the secretion of angiogenic factors

    PubMed Central

    Bijnsdorp, I V; Capriotti, F; Kruyt, F A E; Losekoot, N; Fukushima, M; Griffioen, A W; Thijssen, V L; Peters, G J

    2011-01-01

    Background: Thymidine phosphorylase (TP) is often overexpressed in tumours and has a role in tumour aggressiveness and angiogenesis. Here, we determined whether TP increased tumour invasion and whether TP-expressing cancer cells stimulated angiogenesis. Methods: Angiogenesis was studied by exposing endothelial cells (HUVECs) to conditioned medium (CM) derived from cancer cells with high (Colo320TP1=CT-CM, RT112/TP=RT-CM) and no TP expression after which migration (wound-healing-assay) and invasion (transwell-assay) were determined. The involvement of several angiogenic factors were examined by RT–PCR, ELISA and blocking antibodies. Results: Tumour invasion was not dependent on intrinsic TP expression. The CT-CM and RT-CM stimulated HUVEC-migration and invasion by about 15 and 40%, respectively. Inhibition by 10 μ TPI and 100 μ L-dR, blocked migration and reduced the invasion by 50–70%. Thymidine phosphorylase activity in HUVECs was increased by CT-CM. Reverse transcription-polymerase chain reaction revealed a higher mRNA expression of bFGF (Colo320TP1), IL-8 (RT112/TP) and TNF-α, but not VEGF. Blocking antibodies targeting these factors decreased the migration and invasion that was induced by the CT-CM and RT-CM, except for IL-8 in CT-CM and bFGF in RT-CM. Conclusion: In our cell line panels, TP did not increase the tumour invasion, but stimulated the migration and invasion of HUVECs by two different mechanisms. Hence, TP targeting seems to provide a potential additional strategy in the field of anti-angiogenic therapy. PMID:21386840

  8. Enzymatic characterization of AMP phosphorylase and ribose-1,5-bisphosphate isomerase functioning in an archaeal AMP metabolic pathway.

    PubMed

    Aono, Riku; Sato, Takaaki; Yano, Ayumu; Yoshida, Shosuke; Nishitani, Yuichi; Miki, Kunio; Imanaka, Tadayuki; Atomi, Haruyuki

    2012-12-01

    AMP phosphorylase (AMPpase), ribose-1,5-bisphosphate (R15P) isomerase, and type III ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) have been proposed to constitute a novel pathway involved in AMP metabolism in the Archaea. Here we performed a biochemical examination of AMPpase and R15P isomerase from Thermococcus kodakarensis. R15P isomerase was specific for the α-anomer of R15P and did not recognize other sugar compounds. We observed that activity was extremely low with the substrate R15P alone but was dramatically activated in the presence of AMP. Using AMP-activated R15P isomerase, we reevaluated the substrate specificity of AMPpase. AMPpase exhibited phosphorylase activity toward CMP and UMP in addition to AMP. The [S]-v plot (plot of velocity versus substrate concentration) of the enzyme toward AMP was sigmoidal, with an increase in activity observed at concentrations higher than approximately 3 mM. The behavior of the two enzymes toward AMP indicates that the pathway is intrinsically designed to prevent excess degradation of intracellular AMP. We further examined the formation of 3-phosphoglycerate from AMP, CMP, and UMP in T. kodakarensis cell extracts. 3-Phosphoglycerate generation was observed from AMP alone, and from CMP or UMP in the presence of dAMP, which also activates R15P isomerase. 3-Phosphoglycerate was not formed when 2-carboxyarabinitol 1,5-bisphosphate, a Rubisco inhibitor, was added. The results strongly suggest that these enzymes are actually involved in the conversion of nucleoside monophosphates to 3-phosphoglycerate in T. kodakarensis.

  9. Glucose-derived spiro-isoxazolines are anti-hyperglycemic agents against type 2 diabetes through glycogen phosphorylase inhibition.

    PubMed

    Goyard, David; Kónya, Bálint; Chajistamatiou, Aikaterini S; Chrysina, Evangelia D; Leroy, Jérémy; Balzarin, Sophie; Tournier, Michel; Tousch, Didier; Petit, Pierre; Duret, Cédric; Maurel, Patrick; Somsák, László; Docsa, Tibor; Gergely, Pál; Praly, Jean-Pierre; Azay-Milhau, Jacqueline; Vidal, Sébastien

    2016-01-27

    Glycogen phosphorylase (GP) is a target for the treatment of hyperglycaemia in the context of type 2 diabetes. This enzyme is responsible for the depolymerization of glycogen into glucose thereby affecting the levels of glucose in the blood stream. Twelve new d-glucopyranosylidene-spiro-isoxazolines have been prepared from O-peracylated exo-D-glucals by regio- and stereoselective 1,3-dipolar cycloaddition of nitrile oxides generated in situ by treatment of the corresponding oximes with bleach. This mild and direct procedure appeared to be applicable to a broad range of substrates. The corresponding O-unprotected spiro-isoxazolines were evaluated as glycogen phosphorylase (GP) inhibitors and exhibited IC50 values ranging from 1 to 800 μM. Selected inhibitors were further evaluated in vitro using rat and human hepatocytes and exhibited significant inhibitory properties in the primary cell culture. Interestingly, when tested with human hepatocytes, the tetra-O-acetylated spiro-isoxazoline bearing a 2-naphthyl residue showed a much lower IC50 value (2.5 μM), compared to that of the O-unprotected analog (19.95 μM). The most promising compounds were investigated in Zucker fa/fa rat model in acute and sub-chronic assays and decreased hepatic glucose production, which is known to be elevated in type 2 diabetes. This indicates that glucose-based spiro-isoxazolines can be considered as anti-hyperglycemic agents in the context of type 2 diabetes. PMID:26708111

  10. The Other Double Helix--The Fascinating Chemistry of Starch

    NASA Astrophysics Data System (ADS)

    Hancock, Robert D.; Tarbet, Bryon J.

    2000-08-01

    Current textbooks deal only briefly with the chemistry of starch. A short review with 21 references is presented, describing the structure of starch and indicating the double helix structure of A-type and B-type starch. The structure of the starch granule is examined, pointing out the existence of growth rings of alternating crystalline and noncrystalline starch, with growing amylopectin molecules extending from the hilum (point of origin) to the surface of the starch granule. The swelling of starch granules in water, above the gelatinization temperature of about 60 °C, is discussed. The process of gelatinization involves unraveling of the starch helix and a manyfold increase in volume of the starch granule as water is imbibed and bound to the unraveled starch polymer by hydrogen bonding. Baking bread or pastries causes unraveling of the starch helix, and the process by which these products become stale corresponds primarily to the re-forming of the starch helix. The importance of this phenomenon in food science is discussed. The absorption of nonpolar linear molecules such as I2, or linear nonpolar portions of molecules such as n-butanol or fats and phospholipids, by the C-type helix of starch is examined. The way in which starch is structurally modified to retard staling is discussed in relation to food technology.

  11. Inhibition and Structure of Trichomonas vaginalis Purine Nucleoside Phosphorylase with Picomolar Transition State Analogues

    SciTech Connect

    Rinaldo-Matthis,A.; Wing, C.; Ghanem, M.; Deng, H.; Wu, P.; Gupta, A.; Tyler, P.; Evans, G.; Furneaux, R.; et al.

    2007-01-01

    Trichomonas vaginalis is a parasitic protozoan purine auxotroph possessing a unique purine salvage pathway consisting of a bacterial type purine nucleoside phosphorylase (PNP) and a purine nucleoside kinase. Thus, T. vaginalis PNP (TvPNP) functions in the reverse direction relative to the PNPs in other organisms. Immucillin-A (ImmA) and DADMe-Immucillin-A (DADMe-ImmA) are transition stte mimics of adenosine with geometric and electrostatic features that resemble early and late transition states of adenosine at the transition state stabilized by TvPNP. ImmA demonstrates slow-onset tight-binding inhibition with TvPNP, to give an equilibrium dissociation constant of 87 pM, an inhibitor release half-time of 17.2 min, and a K{sub m}/K{sub d} ratio of 70,100. DADMe-ImmA resembles a late ribooxacarbenium ion transition state for TvPNP to give a dissociation constant of 30 pM, an inhibitor release half-time of 64 min, and a K{sub m}/K{sub d} ratio of 203,300. The tight binding of DADMe-ImmA supports a late S{sub N}1 transition state. Despite their tight binding to TvPNP, ImmA and DADMe-ImmA are weak inhibitors of human and P. falciparum PNPs. The crystal structures of the TvPNP-ImmA{center_dot}PO{sub 4} and TvPNP{center_dot}DADMe-ImmA{center_dot}PO{sub 4} ternary complexes differ from previous structures with substrate anologues. The tight binding with DADMe-ImmA is in part due to a 2.7 {angstrom} ionic interaction between a PO{sub 4} oxygen and the N1 cation of the hydroxypyrrolidine and is weaker in the TvPNP{center_dot}ImmA{center_dot}PO{sub 4} structure at 3.5 {angstrom}. However, the TvPNP{center_dot}ImmA{center_dot}PO{sub 4} structure includes hydrogen bonds between the 2'-hydroxyl and the protein that are not present in TvPNP{center_dot}DADMe-ImmA{center_dot}PO{sub 4}. These structures explain why DADMe-ImmA binds tighter than ImmA. Immucillin-H is a 12 nM inhibitor of TvPNP but a 56 pM inhibitor of human PNP. And this difference is explained by isotope

  12. Effect of waxy rice flour and cassava starch on freeze-thaw stability of rice starch gels.

    PubMed

    Charoenrein, Sanguansri; Preechathammawong, Nutsuda

    2012-10-01

    Repeatedly frozen and thawed rice starch gel affects quality. This study investigated how incorporating waxy rice flour (WF) and cassava starch (CS) in rice starch gel affects factors used to measure quality. When rice starch gels containing 0-2% WF and CS were subjected to 5 freeze-thaw cycles, both WF and CS reduced the syneresis in first few cycles. However CS was more effective in reducing syneresis than WF. The different composite arrangement of rice starch with WF or CS caused different mechanisms associated with the rice starch gel retardation of retrogradation, reduced the spongy structure and lowered syneresis. Both swollen granules of rice starch and CS caused an increase in the hardness of the unfrozen and freeze-thawed starch gel while highly swollen WF granules caused softer gels. These results suggested that WF and CS were effective in preserving quality in frozen rice starch based products.

  13. Production of a freeze-thaw-stable potato starch by antisense inhibition of three starch synthase genes.

    PubMed

    Jobling, Stephen A; Westcott, Roger J; Tayal, Akash; Jeffcoat, Roger; Schwall, Gerhard P

    2002-03-01

    The use of unmodified starches in frozen foods is severely limited by the undesirable textural changes that occur after freezing and thawing. Retrogradation of glucan chains leads to syneresis, a separation of the starch gel and water phases. Stabilization of the starch structure is normally achieved by chemical modification to prevent these changes from occurring. We have now created a freeze-thaw-stable potato starch by alteration of starch composition and structure by genetic modification. An amylose-free starch with short-chain amylopectin was produced by simultaneous antisense downregulation of three starch synthase genes. This starch is extremely freeze-thaw-stable and shows no syneresis even after five freeze-thaw cycles. The use of this starch has potential for environmental and consumer benefits because its production requires no chemical modification.

  14. Resistant Starch and Starch Thermal Characteristics in Exotic Corn Lines Grown in Temperate and Tropical Environments

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Corn as a food that is heated and cooled to allow starch retrogradation has higher levels of resistant starch (RS). Increasing the amount of RS can make corn an even healthier food and may be accomplished by breeding and selection, especially by using exotic germplasm. Sixty breeding lines of introg...

  15. Resistant-starch Formation in High-amylose Maize Starch During Kernel Development

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of this study was to understand the resistant-starch (RS) formation during the kernel development of high-amylose maize, GEMS-0067 line. RS content of the starch, determined using AOAC Method 991.43 for total dietary fiber, increased with kernel maturation and the increase in amylose/...

  16. Physicochemical properties and in vitro starch digestibility of potato starch/protein blends.

    PubMed

    Lu, Zhan-Hui; Donner, Elizabeth; Yada, Rickey Y; Liu, Qiang

    2016-12-10

    This study aimed to investigate effects of starch-protein interactions on physicochemical properties and in vitro starch digestibility of composite potato starch/protein blends (0, 5, 10, or 15% protein) during processing (cooking, cooling and reheating). The effect on recrystallization and short-range ordering in starch was studied by light microscopy, differential scanning calorimetry and Fourier transform infrared spectroscopy. The results show that protein in the blend proportionally restricted starch granule swelling during cooking and facilitated amylopectin recrystallization during cold-storage. The facilitating effect of protein diminished with increasing blend ratio. Resistant starch content in the processed blends was positively correlated to intensity ratio of 1053/1035cm(-1) in FTIR spectra arising from slow retrogradation of amylopectin (r(2)>0.88, P≤0.05), whose formation was favored by the presence of protein in the blends and further enhanced by cooling of cooked blends. As a conclusion, starch-protein interaction reduced starch digestibility of the processed blends. PMID:27577912

  17. Preparation and characterization of resistant starch III from elephant foot yam (Amorphophallus paeonifolius) starch.

    PubMed

    Reddy, Chagam Koteswara; Haripriya, Sundaramoorthy; Noor Mohamed, A; Suriya, M

    2014-07-15

    The purpose of this study was to assess the properties of resistant starch (RS) III prepared from elephant foot yam starch using pullulanase enzyme. Native and gelatinized starches were subjected to enzymatic hydrolysis (pullulanase, 40 U/g per 10h), autoclaved (121°C/30 min), stored under refrigeration (4°C/24h) and then lyophilized. After preparation of resistant starch III, the morphological, physical, chemical and functional properties were assessed. The enzymatic and retrogradation process increased the yield of resistant starch III from starch with a concomitant increase increase in its water absorption capacity and water solubility index. A decrease in swelling power was observed due to the hydrolysis and thermal process. Te reduced pasting properties and hardness of resistant starch III were associated with the disintegration of starch granules due to the thermal process. The viscosity was found to be inversely proportional to the RS content in the sample. The thermal properties of RS increased due to retrogradation and recrystallization (P<0.05). PMID:24594151

  18. Position of modifying groups on starch chains of octenylsuccinic anhydride-modified waxy maize starch

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Octenylsuccinic anhydride (OSA)-modified starches with degree of substitution of 0.018 (OS-S-L) and 0.092 (OS-S-H) were prepared from granular native waxy maize starch in an aqueous slurry system. The substitution distribution of OS groups was investigated by enzyme hydrolysis followed by chromatogr...

  19. Preparation and characterization of resistant starch III from elephant foot yam (Amorphophallus paeonifolius) starch.

    PubMed

    Reddy, Chagam Koteswara; Haripriya, Sundaramoorthy; Noor Mohamed, A; Suriya, M

    2014-07-15

    The purpose of this study was to assess the properties of resistant starch (RS) III prepared from elephant foot yam starch using pullulanase enzyme. Native and gelatinized starches were subjected to enzymatic hydrolysis (pullulanase, 40 U/g per 10h), autoclaved (121°C/30 min), stored under refrigeration (4°C/24h) and then lyophilized. After preparation of resistant starch III, the morphological, physical, chemical and functional properties were assessed. The enzymatic and retrogradation process increased the yield of resistant starch III from starch with a concomitant increase increase in its water absorption capacity and water solubility index. A decrease in swelling power was observed due to the hydrolysis and thermal process. Te reduced pasting properties and hardness of resistant starch III were associated with the disintegration of starch granules due to the thermal process. The viscosity was found to be inversely proportional to the RS content in the sample. The thermal properties of RS increased due to retrogradation and recrystallization (P<0.05).

  20. Water diffusivity in starch-based systems.

    PubMed

    Leslie, R B; Carillo, P J; Chung, T Y; Gilbert, S G; Hayakawa, K; Marousis, S; Saravacos, G D; Solberg, M

    1991-01-01

    The objective of this study was to investigate the influence of structure, and component interactions, on the sorption and transport properties of water in starch-based systems. We compared the effective diffusivity (Deff) of water in two starches, with differing amylose-amylopectin ratios, using either kinetics of water adsorption or analysis of drying curves (water desorption) to estimate Deff. The effect of incorporating small sugar molecules into the granular or gelatinized starch matrices on Deff was measured by drying curve analysis. To investigate the possible mechanisms of water transport, the porosity and microscopic appearance of the samples at different stages of drying were determined. In a complementary study, sorption isotherms and the number of accessible "binding" sites in the starch and starch-sugar systems were determined using gravimetric analysis and inverse gas chromatography (IGC) 'probe analysis'. In the case of the starch-sugar systems, the measurements were made after the components had been 'mechanically mixed', or after more intimate mixing had been achieved by a co-freeze-drying process. The Deff of the starches was found to depend, in a complex way, on the moisture content of the samples. At relatively high moisture contents, the predominant mode of water transport was by liquid diffusion. As the samples became drier, their porosity increased, and the predominant mode of moisture transport was by vapor phase diffusion. As the samples became very dry (less than 10% water content), Deff fell significantly. Incorporation of sugars, in general, led to a reduction of Deff, which was correlated with a corresponding fall in porosity. In agreement with the findings of other workers, for the starches studied, the value of Deff determined from water adsorption measurements was significantly less than Deff determined from water desorption (drying curve analysis). The form of the Deff versus moisture content relationship was, however, independent

  1. Starch phosphorylation in potato tubers is influenced by allelic variation in the genes encoding glucan water dikinase, starch branching enzymes I and II, and starch synthase III

    PubMed Central

    Carpenter, Margaret A.; Joyce, Nigel I.; Genet, Russell A.; Cooper, Rebecca D.; Murray, Sarah R.; Noble, Alasdair D.; Butler, Ruth C.; Timmerman-Vaughan, Gail M.

    2015-01-01

    Starch phosphorylation is an important aspect of plant metabolism due to its role in starch degradation. Moreover, the degree of phosphorylation of starch determines its physicochemical properties and is therefore relevant for industrial uses of starch. Currently, starch is chemically phosphorylated to increase viscosity and paste stability. Potato cultivars with elevated starch phosphorylation would make this process unnecessary, thereby bestowing economic and environmental benefits. Starch phosphorylation is a complex trait which has been previously shown by antisense gene repression to be influenced by a number of genes including those involved in starch synthesis and degradation. We have used an association mapping approach to discover genetic markers associated with the degree of starch phosphorylation. A diverse collection of 193 potato lines was grown in replicated field trials, and the levels of starch phosphorylation at the C6 and C3 positions of the glucosyl residues were determined by mass spectrometry of hydrolyzed starch from tubers. In addition, the potato lines were genotyped by amplicon sequencing and microsatellite analysis, focusing on candidate genes known to be involved in starch synthesis. As potato is an autotetraploid, genotyping included determination of allele dosage. Significant associations (p < 0.001) were found with SNPs in the glucan water dikinase (GWD), starch branching enzyme I (SBEI) and the starch synthase III (SSIII) genes, and with a SSR allele in the SBEII gene. SNPs in the GWD gene were associated with C6 phosphorylation, whereas polymorphisms in the SBEI and SBEII genes were associated with both C6 and C3 phosphorylation and the SNP in the SSIII gene was associated with C3 phosphorylation. These allelic variants have potential as genetic markers for starch phosphorylation in potato. PMID:25806042

  2. Effects of cooking methods and starch structures on starch hydrolysis rates of rice.

    PubMed

    Reed, Michael O; Ai, Yongfeng; Leutcher, Josh L; Jane, Jay-lin

    2013-07-01

    This study aimed to understand effects of different cooking methods, including steamed, pilaf, and traditional stir-fried, on starch hydrolysis rates of rice. Rice grains of 3 varieties, japonica, indica, and waxy, were used for the study. Rice starch was isolated from the grain and characterized. Amylose contents of starches from japonica, indica, and waxy rice were 13.5%, 18.0%, and 0.9%, respectively. The onset gelatinization temperature of indica starch (71.6 °C) was higher than that of the japonica and waxy starch (56.0 and 56.8 °C, respectively). The difference was attributed to longer amylopectin branch chains of the indica starch. Starch hydrolysis rates and resistant starch (RS) contents of the rice varieties differed after they were cooked using different methods. Stir-fried rice displayed the least starch hydrolysis rate followed by pilaf rice and steamed rice for each rice variety. RS contents of freshly steamed japonica, indica, and waxy rice were 0.7%, 6.6%, and 1.3%, respectively; those of rice pilaf were 12.1%, 13.2%, and 3.4%, respectively; and the stir-fried rice displayed the largest RS contents of 15.8%, 16.6%, and 12.1%, respectively. Mechanisms of the large RS contents of the stir-fried rice were studied. With the least starch hydrolysis rate and the largest RS content, stir-fried rice would be a desirable way of preparing rice for food to reduce postprandial blood glucose and insulin responses and to improve colon health of humans.

  3. Polysaccharide fraction from higher plants which strongly interacts with the cytosolic phosphorylase isozyme. I. Isolation and characterization. [Spinacia oleracea L. ; Pisum sativum L

    SciTech Connect

    Yang, Yi; Steup, M. )

    1990-11-01

    From leaves of Spinacia oleracea L. or from Pisum sativum L. and from cotyledons of germinating pea seeds a high molecular weight polysaccharide fraction was isolated. The apparent size of the fraction, as determined by gel filtration, was similar to that of dextran blue. Following acid hydrolysis the monomer content of the polysaccharide preparation was studied using high pressure liquid and thin layer chromatography. Glucose, galactose, arabinose, and ribose were the main monosaccharide compounds. The native polysaccharide preparation interacted strongly with the cytosolic isozyme of phosphorylase (EC 2.4.1.1). Interaction with the plastidic phosphorylase isozyme(s) was by far weaker. Interaction with the cytosolic isozyme was demonstrated by affinity electrophoresis, kinetic measurements, and by {sup 14}C-labeling experiments in which the glucosyl transfer from ({sup 14}C)glucose 1-phosphate to the polysaccharide preparation was monitored.

  4. Starches of varied digestibilities differentially modify intestinal function in rats.

    PubMed

    Lajvardi, A; Mazarin, G I; Gillespie, M B; Satchithanandam, S; Calvert, R J

    1993-12-01

    Starches of different digestibilities may enter the colon to different extents and alter colonic function. Male Fischer 344 rats were fed diets containing 25% cooked potato starch, arrowroot starch, high amylose cornstarch or raw potato starch for 6 wk. Fecal weight, transit time, colonic thymidine kinase activity (a marker for cell proliferation), and weight, starch content and pH of the cecum and proximal and distal colon were measured. Raw potato starch was much less completely digested than high amylose cornstarch, resulting in a 32-fold greater amount of undigested starch entering the cecum in the raw potato starch group. Both the high amylose cornstarch and raw potato starch diets significantly enhanced fecal weight and produced large intestinal hypertrophy, effects that were greatest in the raw potato starch group. Raw potato starch feeding was associated with the highest level of thymidine kinase activity, although the differences in thymidine kinase activity among the four groups were not significant. This diet also produced a 50% longer transit time. Entry of a large amount of raw potato starch into the colon resulted in greater luminal acidity, greater luminal bulk and slower transit. A much smaller amount of starch entered the colon in the high amylose cornstarch group and resulted in fecal bulking but no alteration in transit. PMID:8263598

  5. Starches of varied digestibilities differentially modify intestinal function in rats.

    PubMed

    Lajvardi, A; Mazarin, G I; Gillespie, M B; Satchithanandam, S; Calvert, R J

    1993-12-01

    Starches of different digestibilities may enter the colon to different extents and alter colonic function. Male Fischer 344 rats were fed diets containing 25% cooked potato starch, arrowroot starch, high amylose cornstarch or raw potato starch for 6 wk. Fecal weight, transit time, colonic thymidine kinase activity (a marker for cell proliferation), and weight, starch content and pH of the cecum and proximal and distal colon were measured. Raw potato starch was much less completely digested than high amylose cornstarch, resulting in a 32-fold greater amount of undigested starch entering the cecum in the raw potato starch group. Both the high amylose cornstarch and raw potato starch diets significantly enhanced fecal weight and produced large intestinal hypertrophy, effects that were greatest in the raw potato starch group. Raw potato starch feeding was associated with the highest level of thymidine kinase activity, although the differences in thymidine kinase activity among the four groups were not significant. This diet also produced a 50% longer transit time. Entry of a large amount of raw potato starch into the colon resulted in greater luminal acidity, greater luminal bulk and slower transit. A much smaller amount of starch entered the colon in the high amylose cornstarch group and resulted in fecal bulking but no alteration in transit.

  6. Microwave-assisted synthesis of C-8 aryl and heteroaryl inosines and determination of their inhibitory activities against Plasmodium falciparum purine nucleoside phosphorylase.

    PubMed

    Gigante, Alba; Priego, Eva-María; Sánchez-Carrasco, Paula; Ruiz-Pérez, Luis Miguel; Vande Voorde, Johan; Camarasa, María-José; Balzarini, Jan; González-Pacanowska, Dolores; Pérez-Pérez, María-Jesús

    2014-07-23

    8-Arylinosines have been scarcely studied for therapeutic purposes, probably due to difficulties in their synthesis. The recently described direct arylation reaction at position 8 of purine nucleosides has been employed to synthesize a series of 8-aryl and 8-pyridylinosines. These compounds have been studied for hydrolytic stability and subjected to biological evaluation. Three compounds have shown a pronounced specific inhibition of Plasmodium falciparum-encoded purine nucleoside phosphorylase, an important target for antimalarial chemotherapy. PMID:24929343

  7. Anthranilimide based glycogen phosphorylase inhibitors for the treatment of type 2 diabetes. Part 3: X-ray crystallographic characterization, core and urea optimization and in vivo efficacy

    SciTech Connect

    Thomson, Stephen A.; Banker, Pierette; Bickett, D. Mark; Boucheron, Joyce A.; Carter, H. Luke; Clancy, Daphne C.; Cooper, Joel P.; Dickerson, Scott H.; Garrido, Dulce M.; Nolte, Robert T.; Peat, Andrew J.; Sheckler, Lauren R.; Sparks, Steven M.; Tavares, Francis X.; Wang, Liping; Wang, Tony Y.; Weiel, James E.

    2009-05-15

    Key binding interactions of the anthranilimide based glycogen phosphorylase a (GPa) inhibitor 2 from X-ray crystallography studies are described. This series of compounds bind to the AMP site of GP. Using the binding information the core and the phenyl urea moieties were optimized. This work culminated in the identification of compounds with single nanomolar potency as well as in vivo efficacy in a diabetic model.

  8. Concerted suppression of all starch branching enzyme genes in barley produces amylose-only starch granules

    PubMed Central

    2012-01-01

    Background Starch is stored in higher plants as granules composed of semi-crystalline amylopectin and amorphous amylose. Starch granules provide energy for the plant during dark periods and for germination of seeds and tubers. Dietary starch is also a highly glycemic carbohydrate being degraded to glucose and rapidly absorbed in the small intestine. But a portion of dietary starch, termed “resistant starch” (RS) escapes digestion and reaches the large intestine, where it is fermented by colonic bacteria producing short chain fatty acids (SCFA) which are linked to several health benefits. The RS is preferentially derived from amylose, which can be increased by suppressing amylopectin synthesis by silencing of starch branching enzymes (SBEs). However all the previous works attempting the production of high RS crops resulted in only partly increased amylose-content and/or significant yield loss. Results In this study we invented a new method for silencing of multiple genes. Using a chimeric RNAi hairpin we simultaneously suppressed all genes coding for starch branching enzymes (SBE I, SBE IIa, SBE IIb) in barley (Hordeum vulgare L.), resulting in production of amylose-only starch granules in the endosperm. This trait was segregating 3:1. Amylose-only starch granules were irregularly shaped and showed peculiar thermal properties and crystallinity. Transgenic lines retained high-yield possibly due to a pleiotropic upregualtion of other starch biosynthetic genes compensating the SBEs loss. For gelatinized starch, a very high content of RS (65 %) was observed, which is 2.2-fold higher than control (29%). The amylose-only grains germinated with same frequency as control grains. However, initial growth was delayed in young plants. Conclusions This is the first time that pure amylose has been generated with high yield in a living organism. This was achieved by a new method of simultaneous suppression of the entire complement of genes encoding starch branching enzymes. We

  9. Three-dimensional structures of unligated uridine phosphorylase from Yersinia pseudotuberculosis at 1.4 Å resolution and its complex with an antibacterial drug

    NASA Astrophysics Data System (ADS)

    Balaev, V. V.; Lashkov, A. A.; Gabdulkhakov, A. G.; Dontsova, M. V.; Mironov, A. S.; Betzel, C.; Mikhailov, A. M.

    2015-07-01

    Uridine phosphorylases play an essential role in the cellular metabolism of some antibacterial agents. Acute infectious diseases (bubonic plague, yersiniosis, pseudotuberculosis, etc., caused by bacteria of the genus Yersinia) are treated using both sulfanilamide medicines and antibiotics, including trimethoprim. The action of an antibiotic on a bacterial cell is determined primarily by the character of its interactions with cellular components, including those which are not targets (for example, with pyrimidine phosphorylases). This type of interaction should be taken into account in designing drugs. The three-dimensional structure of uridine phosphorylase from the bacterium Yersinia pseudotuberculosis ( YptUPh) with the free active site was determined for the first time by X-ray crystallography and refined at 1.40 Å resolution (DPI = 0.062 Å; ID PDB: 4OF4). The structure of the complex of YptUPh with the bacteriostatic drug trimethoprim was studied by molecular docking and molecular dynamics methods. The trimethoprim molecule was shown to be buffered by the enzyme YptUPh, resulting in a decrease in the efficiency of the treatment of infectious diseases caused by bacteria of the genus Yersinia with trimethoprim.

  10. Effects of eugenol-reduced clove extract on glycogen phosphorylase b and the development of diabetes in db/db mice.

    PubMed

    Sanae, Fujiko; Kamiyama, Ogusa; Ikeda-Obatake, Kyoko; Higashi, Yasuhiko; Asano, Naoki; Adachi, Isao; Kato, Atsushi

    2014-02-01

    We found that the 50% aqueous EtOH extract of clove (Syzygium aromaticum) had potent dose-dependent inhibitory activity toward glycogen phosphorylase b and glucagon-stimulated glucose production in primary rat hepatocytes. Among the components, eugeniin inhibited glycogen phosphorylase b and glucagon-stimulated glucose production in primary rat hepatocytes, with IC50 values of 0.14 and 4.7 μM, respectively. In sharp contrast, eugenol showed no significant inhibition toward glycogen phosphorylase b, even at a concentration of 400 μM. Eugenol-reduced clove extracts (erCE) were prepared and when fed to a db/db mouse they clearly suppressed the blood glucose and HbA1c levels. Furthermore, plasma triglyceride and non-esterified fatty acid levels in 5% and 10% erCE-fed db/db mice were significantly lowered, compared with control db/db mice without erCE supplementation. These results suggested that dietary supplementation with the erCE could beneficially modify glucose and lipid metabolism and contribute to the prevention of the progress of hyperglycemia and metabolic syndrome.

  11. Reaction of phosphorylase-a with α-D-glucose 1-phosphate and maltodextrin acceptors to give products with degree of polymerization 6-89.

    PubMed

    Kazłowski, Bartosz; Ko, Yuan-Tih

    2014-06-15

    A series of linear glucan saccharides (GS) with defined quantity and degree of polymerization (DP) were synthesized from α-d-glucose 1-phosphate (α-d-Glc 1-P) by phosphorylase-a. The GS product fractions with average DP 11, 22, 38, 52, 60, 70, and 79 were measured by HPSEC-ELSD system. Then the same seven fractions were resolved into individual peaks with DP: 6-14, 10-32, 27-55, 37-67, 44-75, 49-83 and 53-89 by HPAEC-PAD system. Results showed that measurement of α-d-Glc 1-P amount consuming during GS synthesis by both systems enable calculation of reaction yield. The reaction yield for the 24h biosynthesis of the GS product was 25.3% (measured by HPSEC-ELSD) or 29.1% (measured by HPAEC-PAD). The HPSEC-ELSD and HPAEC-PAD systems were also successfully used for phosphorylase-a activity measurement in order to perform its kinetic characterization. This study established feasible systems for preparation of various sizes of the GS with defined DP and quantity as well as characterization of phosphorylase-a kinetics.

  12. Initiator Effects in Reactive Extrusion of Starch Graft Copolymers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Graft copolymers of starch with water-soluble polymers such as polyacrylamide have potential applications including hydrogels, superabsorbents, and thickening agents. Reactive extrusion is a rapid, continuous method for production of starch graft copolymers with high reaction and grafting efficienc...

  13. Starch: its metabolism, evolution, and biotechnological modification in plants.

    PubMed

    Zeeman, Samuel C; Kossmann, Jens; Smith, Alison M

    2010-01-01

    Starch is the most widespread and abundant storage carbohydrate in plants. We depend upon starch for our nutrition, exploit its unique properties in industry, and use it as a feedstock for bioethanol production. Here, we review recent advances in research in three key areas. First, we assess progress in identifying the enzymatic machinery required for the synthesis of amylopectin, the glucose polymer responsible for the insoluble nature of starch. Second, we discuss the pathways of starch degradation, focusing on the emerging role of transient glucan phosphorylation in plastids as a mechanism for solubilizing the surface of the starch granule. We contrast this pathway in leaves with the degradation of starch in the endosperm of germinated cereal seeds. Third, we consider the evolution of starch biosynthesis in plants from the ancestral ability to make glycogen. Finally, we discuss how this basic knowledge has been utilized to improve and diversify starch crops.

  14. Functionality of porous starch obtained by amylase or amyloglucosidase treatments.

    PubMed

    Dura, A; Błaszczak, W; Rosell, C M

    2014-01-30

    Porous starch is attracting very much attention for its absorption and shielding ability in many food applications. The effect of two different enzymes, fungal α-amylase (AM) or amyloglucosidase (AMG), on corn starch at sub-gelatinization temperature was studied as an alternative to obtain porous starch. Biochemical features, thermal and structural analyses of treated starches were studied. Microscopic analysis of the granules confirmed the enzymatic modification of the starches obtaining porous structures with more agglomerates in the case of AMG treated starches. Several changes in thermal properties and hydrolysis kinetics were observed in enzymatically modified starches. Hydration properties were significantly affected by enzymatic modification being greater influenced by AMG activity, and the opposite trend was observed in the pasting properties. Overall, results showed that enzymatic modification at sub-gelatinization temperatures really offer an attractive alternative for obtaining porous starch granules to be used in a variety of foods applications.

  15. Regulation of starch synthesis in potato tubers

    SciTech Connect

    Davies, H.; Oparka, K.; Viola, R.; Wright, K.; Ross, H. )

    1990-05-01

    Following tuber excision from the mother plant sucrose synthase activity fell from 3,120 to 960 nmol/g.f. wt./h within 7 days and starch synthesis ({sup 14}C sucrose incorporated into isolated discs) from 23 to 7 nmol/g.f. wt./h. While the maximum catalytic activity of sucrose synthase was more than sufficient to account for the observed rate of starch synthesis a maximum of 27% of sucrose incorporated by discs was converted into starch within 3 h. This compared with 80% conversion of {sup 14}C glucose incorporated. Tuber excision also reduced the rate of starch biosynthesis with glucose as a substrate (from 206 to 64 nmol/g.f. wt./h). The activities of UDPG-pyrophosphorylase, PPi-PFK, ATP-PFK, starch synthase and hexokinase (glucose or fructose substrates) were unaffected by tuber removal. ADPG pyrophosphorylase activity was reduced from 8,000 to 4,500 nmol/g.f. wt./h. Preliminary experiments indicate that the decline in sucrose synthease activity is prevented by maintaining sucrose flux into tubers through the cut stolon.

  16. Probing starch-iodine interaction by atomic force microscopy.

    PubMed

    Du, Xiongwei; An, Hongjie; Liu, Zhongdong; Yang, Hongshun; Wei, Lijuan

    2014-01-01

    We explored the interaction of iodine with three crystalline type starches, corn, potato, and sweet potato starches using atomic force microscopy. Results revealed that starch molecules aggregated through interaction with iodine solution as well as iodine vapor. Detailed fine structures such as networks, chains, and super-helical structures were found in iodide solution tests. The nanostructures formed due to iodine adsorption could help to understand the formation and properties of the starch-iodine complex.

  17. Probing starch-iodine interaction by atomic force microscopy.

    PubMed

    Du, Xiongwei; An, Hongjie; Liu, Zhongdong; Yang, Hongshun; Wei, Lijuan

    2014-01-01

    We explored the interaction of iodine with three crystalline type starches, corn, potato, and sweet potato starches using atomic force microscopy. Results revealed that starch molecules aggregated through interaction with iodine solution as well as iodine vapor. Detailed fine structures such as networks, chains, and super-helical structures were found in iodide solution tests. The nanostructures formed due to iodine adsorption could help to understand the formation and properties of the starch-iodine complex. PMID:24338992

  18. Production of resistant starch by extrusion cooking of acid-modified normal-maize starch.

    PubMed

    Hasjim, Jovin; Jane, Jay-Lin

    2009-09-01

    The objective of this study was to utilize extrusion cooking and hydrothermal treatment to produce resistant starch (RS) as an economical alternative to a batch-cooking process. A hydrothermal treatment (110 degrees C, 3 d) of batch-cooked and extruded starch samples facilitated propagation of heat-stable starch crystallites and increased the RS contents from 2.1% to 7.7% up to 17.4% determined using AOAC Method 991.43 for total dietary fiber. When starch samples were batch cooked and hydrothermally treated at a moisture content below 70%, acid-modified normal-maize starch (AMMS) produced a greater RS content than did native normal-maize starch (NMS). This was attributed to the partially hydrolyzed, smaller molecules in the AMMS, which had greater mobility and freedom than the larger molecules in the NMS. The RS contents of the batch-cooked and extruded AMMS products after the hydrothermal treatment were similar. A freezing treatment of the AMMS samples at -20 degrees C prior to the hydrothermal treatment did not increase the RS content. The DSC thermograms and the X-ray diffractograms showed that retrograded amylose and crystalline starch-lipid complex, which had melting temperatures above 100 degrees C, accounted for the RS contents.

  19. Association mapping of starch physicochemical properties with starch biosynthesizing genes in waxy rice (Oryza sativa L.).

    PubMed

    Xu, Feifei; Zhang, Gan; Tong, Chuan; Sun, Xiao; Corke, Harold; Sun, Mei; Bao, Jinsong

    2013-10-23

    Waxy (glutinous) rice is widely used in traditional foods, and understanding the genetic bases of its diverse physicochemical properties will contribute to breeding of new waxy rice with unique qualities. The objective of this study was to investigate the genetic relationship between the starch biosynthesis related genes and the physicochemical properties of waxy rice using association mapping. A total of 36 molecular markers representing 18 genes were used to genotype 50 waxy rice accessions for which starch properties were previously available. Most of the starch properties differed between high and low gelatinization temperature (GT) groups, whereas most traits were similar between the low-GT indica rice and low-GT japonica rice, suggesting GT was the main determinant of the starch quality of waxy rice. Association mapping indicated that the starch properties of waxy rice were mainly controlled by starch synthase IIa (SSIIa or SSII-3, a major gene responsible for the gelatinization temperature) and SSI. It was found that gene-gene interactions were also important for the genetic control of starch properties of waxy rice. This study suggests that application of the functional SNPs of SSIIa in molecular breeding may facilitate quality improvement of waxy rice.

  20. Starch behaviors and mechanical properties of starch blend films with different plasticizers.

    PubMed

    Nguyen Vu, Hoang Phuong; Lumdubwong, Namfone

    2016-12-10

    The main objective of the study was to gain insight into structural and mechanical starch behaviors of the plasticized starch blend films. Mechanical properties and starch behaviors of cassava (CS)/and mungbean (MB) (50/50, w/w) starch blend films containing glycerol (Gly) or sorbitol (Sor) at 33% weight content were investigated. It was found that tensile strength TS and %E of the Gly-CSMB films were similar to those of MB films; but%E of all Sor-films was identical. TS of plasticized films increased when AM content and crystallinity increased. When Sor was substituted for Gly, crystallinity of starch films and their TS increased. The CSMB and MB films had somewhat a similar molecular profile and comparable mechanical properties. Therefore, it was proposed the starch molecular profile containing amylopectin with high M¯w, low M¯w of amylose, and the small size of intermediates may impart the high TS and%E of starch films. PMID:27577902

  1. Morphological, Thermal, and Rheological Properties of Starches from Maize Mutants Deficient in Starch Synthase III.

    PubMed

    Zhu, Fan; Bertoft, Eric; Li, Guantian

    2016-08-31

    Morphological, thermal, and rheological properties of starches from maize mutants deficient in starch synthase III (SSIII) with a common genetic background (W64A) were studied and compared with the wild type. SSIII deficiency reduced granule size of the starches from 16.7 to ∼11 μm (volume-weighted mean). Thermal analysis showed that SSIII deficiency decreased the enthalpy change of starch during gelatinization. Steady shear analysis showed that SSIII deficiency decreased the consistency coefficient and yield stress during steady shearing, whereas additional deficiency in granule-bound starch synthase (GBSS) increased these values. Dynamic oscillatory analysis showed that SSIII deficiency decreased G' at 90 °C during heating and increased it when the paste was cooled to 25 °C at 40 Hz during a frequency sweep. Additional GBSS deficiency further decreased the G'. Structural and compositional bases responsible for these changes in physical properties of the starches are discussed. This study highlighted the relationship between SSIII and some physicochemical properties of maize starch. PMID:27523327

  2. Measurement of resistant starch by enzymatic digestion in starch and selected plant materials: collaborative study.

    PubMed

    McCleary, Barry V; McNally, Marian; Rossiter, Patricia

    2002-01-01

    Interlaboratory performance statistics was determined for a method developed to measure the resistant starch (RS) content of selected plant food products and a range of commercial starch samples. Food materials examined contained RS (cooked kidney beans, green banana, and corn flakes) and commercial starches, most of which naturally contain, or were processed to yield, elevated RS levels. The method evaluated was optimized to yield RS values in agreement with those reported for in vivo studies. Thirty-seven laboratories tested 8 pairs of blind duplicate starch or plant material samples with RS values between 0.6 (regular maize starch) and 64% (fresh weight basis). For matrixes excluding regular maize starch, repeatability relative standard deviation (RSDr) values ranged from 1.97 to 4.2%, and reproducibility relative standard deviation (RSDR) values ranged from 4.58 to 10.9%. The range of applicability of the test is 2-64% RS. The method is not suitable for products with <1% RS (e.g., regular maize starch; 0.6% RS). For such products, RSDr and RSDR values are unacceptably high. PMID:12374410

  3. Acetylated adipate of retrograded starch as RS 3/4 type resistant starch.

    PubMed

    Kapelko-Żeberska, M; Zięba, T; Spychaj, R; Gryszkin, A

    2015-12-01

    This study was aimed at producing acetylated adipate of retrograded starch (ADA-R) with various degrees of substitution with functional groups and at determining the effect of esterification degree on resistance and pasting characteristics of the produced preparations. Paste was prepared from native potato starch, and afterwards frozen and defrosted. After drying and disintegration, the paste was acetylated and crosslinked using various doses of reagents. An increase in the total degree of esterification of the produced ADA-R-preparation caused an increase in its resistance to the action of amyloglucosidase. Viscosity of the paste produced from ADA-R-preparation in a wide range of acetylation degrees was increasing along with increasing crosslinking of starch. The study demonstrated that acetylated adipate of retrograded starch may be classified as a preparation of RS 3/4 type resistant starch (retrograded starch/chemically-modified starch) with good texture-forming properties. The conducted modification offers the possibility of modeling the level of resistance of the produced preparation.

  4. Acetylated adipate of retrograded starch as RS 3/4 type resistant starch.

    PubMed

    Kapelko-Żeberska, M; Zięba, T; Spychaj, R; Gryszkin, A

    2015-12-01

    This study was aimed at producing acetylated adipate of retrograded starch (ADA-R) with various degrees of substitution with functional groups and at determining the effect of esterification degree on resistance and pasting characteristics of the produced preparations. Paste was prepared from native potato starch, and afterwards frozen and defrosted. After drying and disintegration, the paste was acetylated and crosslinked using various doses of reagents. An increase in the total degree of esterification of the produced ADA-R-preparation caused an increase in its resistance to the action of amyloglucosidase. Viscosity of the paste produced from ADA-R-preparation in a wide range of acetylation degrees was increasing along with increasing crosslinking of starch. The study demonstrated that acetylated adipate of retrograded starch may be classified as a preparation of RS 3/4 type resistant starch (retrograded starch/chemically-modified starch) with good texture-forming properties. The conducted modification offers the possibility of modeling the level of resistance of the produced preparation. PMID:26041205

  5. Physical characterisation of high amylose maize starch and acylated high amylose maize starches.

    PubMed

    Lim, Ya-Mei; Hoobin, Pamela; Ying, DanYang; Burgar, Iko; Gooley, Paul R; Augustin, Mary Ann

    2015-03-01

    The particle size, water sorption properties and molecular mobility of high amylose maize starch (HAMS) and high amylose maize starch acylated with acetate (HAMSA), propionate (HAMSP) and butyrate (HAMSB) were investigated. Acylation increased the mean particle size (D(4,3)) and lowered the specific gravity (G) of the starch granules with an inverse relationship between the length of the fatty acid chain and particle size. Acylation of HAMS with fatty acids lowered the monolayer moisture content with the trend being HAMSBstarch granules by NMR spectroscopy with Carr-Purcell-Meiboom-Gill (CMPG) experiments showed that T2 long was reduced in acylated starches and that drying and storage of the starch granules further reduced T2 long. Analysis of the Free Induction Decay (FID) focussing on the short components of T2 (correlated to the solid matrix), indicated that drying and subsequent storage resulted in alterations of starch at 0.33a(w) and that these changes were reduced with acylation. In vitro enzymatic digestibility of heated starch dispersions by bacterial α-amylase was increased by acylation (HAMSstarch. PMID:25498636

  6. SCANDIUM TRIFLATE CATALYZED ACETYLATION OF STARCH UNDER MILD CONDITIONS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Scandium (III) trifluoromethan sulfonate (Sc(OTf)3) was investigated as a catalyst for the acetylation of starch in order to determine the potential for preparing new types of starch esters under mild conditions. At room temperature, dry granular corn starch reacts with acetic anhydride in the pres...

  7. Adsorption of Polyethylene from Solution onto Starch Film Surfaces

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Starch films were prepared by jet cooking aqueous dispersions of high-amylose starch and then allowing the jet cooked dispersions to air-dry on Teflon surfaces. When the starch films were immersed in 1 % solutions of PE in 1-dodecanol, dodecane and xylene at 120º C and the solutions were allowed to...

  8. Starch as a feedstock for bioproducts and packaging

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Much progress has been achieved in developing starch-based feedstocks as a partial replacement for petroleum-based feedstocks. Although starch remains a poor direct substitute for plastics, composite starch-based materials have useful functional properties and are in commercial production as a repla...

  9. Starch-based Foam Composite Materials: processing and bioproducts

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Starch is an abundant, biodegradable, renewable and low-cost commodity that has been explored as a replacement for petroleum-based plastics. By itself, starch is a poor replacement for plastics because of its moisture sensitivity and brittle properties. Efforts to improve starch properties and funct...

  10. Shape memory starch-clay bionanocomposites.

    PubMed

    Coativy, Gildas; Gautier, Nicolas; Pontoire, Bruno; Buléon, Alain; Lourdin, Denis; Leroy, Eric

    2015-02-13

    1-10% starch/clay bionanocomposites with shape memory properties were obtained by melt processing. X-ray diffraction (XRD) and TEM evidenced the presence of a major fraction of clay tactoids, consisting of 4-5 stacked crystalline layers, with a thickness of 6.8 nm. A significant orientation of the nanoparticles induced by extrusion was also observed. Tensile tests performed above the glass transition of the materials showed that the presence of clay nanoparticles leads to higher elastic modulus and maximum stress, without significant loss in elongation at break which typically reached 100%. Samples submitted to a 50% elongation and cooled below the glass transition showed shape memory behavior. Like unreinforced starch, the bionanocomposites showed complete shape recovery in unconstrained conditions. In mechanically constrained conditions, the maximum recovered stress was significantly improved for the bionanocomposites compared to unreinforced starch, opening promising perspectives for the design of sensors and actuators. PMID:25458305

  11. Retention of Cationic Starch onto Cellulose Fibres

    NASA Astrophysics Data System (ADS)

    Missaoui, Mohamed; Mauret, Evelyne; Belgacem, Mohamed Naceur

    2008-08-01

    Three methods of cationic starch titration were used to quantify its retention on cellulose fibres, namely: (i) the complexation of CS with iodine and measurement of the absorbency of the ensuing blue solution by UV-vis spectroscopy; (ii) hydrolysis of the starch macromolecules followed by the conversion of the resulting sugars to furan-based molecules and quantifying the ensuing mixture by measuring their absorbance at a Ι of 490 nm, using the same technique as previous one and; finally (iii) hydrolysis of starch macromolecules by trifluoro-acetic acid and quantification of the sugars in the resulting hydrolysates by high performance liquid chromatography. The three methods were found to give similar results within the range of CS addition from 0 to 50 mg per g of cellulose fibres.

  12. Starch: Structure, Properties, Chemistry, and Enzymology

    NASA Astrophysics Data System (ADS)

    Robyt, John F.

    Starch is a very important and widely distributed natural product, occurring in the leaves of green plants, seeds, fruits, stems, roots, and tubers. It serves as the chemical storage form of the energy of the sun and is the primary source of energy for the organisms on the Earth. Starch is composed of two kinds of polysaccharides, amylose and amylopectin, exclusively composed of D-glucose residues with α-(1→4) linkages in a linear amylose and α-(1→4) linkages and ˜5% α-(1→6) branch linkages in amylopectin, both combined in a water-insoluble granule that is partially crystalline and whose size, shape, and morphology are dependent on its biological source. The properties, isolation, fractionation, enzymatic degradation, biosynthesis, chemical modification, and specific methods of analysis of starch are presented.

  13. Starch Granule Re-Structuring by Starch Branching Enzyme and Glucan Water Dikinase Modulation Affects Caryopsis Physiology and Metabolism.

    PubMed

    Shaik, Shahnoor S; Obata, Toshihiro; Hebelstrup, Kim H; Schwahn, Kevin; Fernie, Alisdair R; Mateiu, Ramona V; Blennow, Andreas

    2016-01-01

    Starch is of fundamental importance for plant development and reproduction and its optimized molecular assembly is potentially necessary for correct starch metabolism. Re-structuring of starch granules in-planta can therefore potentially affect plant metabolism. Modulation of granule micro-structure was achieved by decreasing starch branching and increasing starch-bound phosphate content in the barley caryopsis starch by RNAi suppression of all three Starch Branching Enzyme (SBE) isoforms or overexpression of potato Glucan Water Dikinase (GWD). The resulting lines displayed Amylose-Only (AO) and Hyper-Phosphorylated (HP) starch chemotypes, respectively. We studied the influence of these alterations on primary metabolism, grain composition, starch structural features and starch granule morphology over caryopsis development at 10, 20 and 30 days after pollination (DAP) and at grain maturity. While HP showed relatively little effect, AO showed significant reduction in starch accumulation with re-direction to protein and β-glucan (BG) accumulation. Metabolite profiling indicated significantly higher sugar accumulation in AO, with re-partitioning of carbon to accumulate amino acids, and interestingly it also had high levels of some important stress-related metabolites and potentially protective metabolites, possibly to elude deleterious effects. Investigations on starch molecular structure revealed significant increase in starch phosphate and amylose content in HP and AO respectively with obvious differences in starch granule morphology at maturity. The results demonstrate that decreasing the storage starch branching resulted in metabolic adjustments and re-directions, tuning to evade deleterious effects on caryopsis physiology and plant performance while only little effect was evident by increasing starch-bound phosphate as a result of overexpressing GWD.

  14. Starch Granule Re-Structuring by Starch Branching Enzyme and Glucan Water Dikinase Modulation Affects Caryopsis Physiology and Metabolism.

    PubMed

    Shaik, Shahnoor S; Obata, Toshihiro; Hebelstrup, Kim H; Schwahn, Kevin; Fernie, Alisdair R; Mateiu, Ramona V; Blennow, Andreas

    2016-01-01

    Starch is of fundamental importance for plant development and reproduction and its optimized molecular assembly is potentially necessary for correct starch metabolism. Re-structuring of starch granules in-planta can therefore potentially affect plant metabolism. Modulation of granule micro-structure was achieved by decreasing starch branching and increasing starch-bound phosphate content in the barley caryopsis starch by RNAi suppression of all three Starch Branching Enzyme (SBE) isoforms or overexpression of potato Glucan Water Dikinase (GWD). The resulting lines displayed Amylose-Only (AO) and Hyper-Phosphorylated (HP) starch chemotypes, respectively. We studied the influence of these alterations on primary metabolism, grain composition, starch structural features and starch granule morphology over caryopsis development at 10, 20 and 30 days after pollination (DAP) and at grain maturity. While HP showed relatively little effect, AO showed significant reduction in starch accumulation with re-direction to protein and β-glucan (BG) accumulation. Metabolite profiling indicated significantly higher sugar accumulation in AO, with re-partitioning of carbon to accumulate amino acids, and interestingly it also had high levels of some important stress-related metabolites and potentially protective metabolites, possibly to elude deleterious effects. Investigations on starch molecular structure revealed significant increase in starch phosphate and amylose content in HP and AO respectively with obvious differences in starch granule morphology at maturity. The results demonstrate that decreasing the storage starch branching resulted in metabolic adjustments and re-directions, tuning to evade deleterious effects on caryopsis physiology and plant performance while only little effect was evident by increasing starch-bound phosphate as a result of overexpressing GWD. PMID:26891365

  15. Starch Granule Re-Structuring by Starch Branching Enzyme and Glucan Water Dikinase Modulation Affects Caryopsis Physiology and Metabolism

    PubMed Central

    Shaik, Shahnoor S.; Obata, Toshihiro; Hebelstrup, Kim H.; Schwahn, Kevin; Fernie, Alisdair R.; Mateiu, Ramona V.; Blennow, Andreas

    2016-01-01

    Starch is of fundamental importance for plant development and reproduction and its optimized molecular assembly is potentially necessary for correct starch metabolism. Re-structuring of starch granules in-planta can therefore potentially affect plant metabolism. Modulation of granule micro-structure was achieved by decreasing starch branching and increasing starch-bound phosphate content in the barley caryopsis starch by RNAi suppression of all three Starch Branching Enzyme (SBE) isoforms or overexpression of potato Glucan Water Dikinase (GWD). The resulting lines displayed Amylose-Only (AO) and Hyper-Phosphorylated (HP) starch chemotypes, respectively. We studied the influence of these alterations on primary metabolism, grain composition, starch structural features and starch granule morphology over caryopsis development at 10, 20 and 30 days after pollination (DAP) and at grain maturity. While HP showed relatively little effect, AO showed significant reduction in starch accumulation with re-direction to protein and β-glucan (BG) accumulation. Metabolite profiling indicated significantly higher sugar accumulation in AO, with re-partitioning of carbon to accumulate amino acids, and interestingly it also had high levels of some important stress-related metabolites and potentially protective metabolites, possibly to elude deleterious effects. Investigations on starch molecular structure revealed significant increase in starch phosphate and amylose content in HP and AO respectively with obvious differences in starch granule morphology at maturity. The results demonstrate that decreasing the storage starch branching resulted in metabolic adjustments and re-directions, tuning to evade deleterious effects on caryopsis physiology and plant performance while only little effect was evident by increasing starch-bound phosphate as a result of overexpressing GWD. PMID:26891365

  16. Morphological and mechanical characterization of thermoplastic starch and its blends with polylactic acid using cassava starch and bagasse

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study aims the use of an agro waste coming from the industrialization of cassava starch, known as cassava bagasse (BG). This material contains residual starch and cellulose fibers which can be used to obtain thermoplastic starch (TPS) and /or blends reinforced with fibers. In this context, it w...

  17. Characterization of Maize Amylose-Extender (ae) Mutant Starches. Part I: Relationship Between Resistant Starch Contents and Molecular Structures

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Endosperm starches were isolated from kernels of seven maize amylose-extender (ae) lines. The resistant starch (RS) contents, measured using AOAC method 991.43, showed that three new ae-mutant starch lines developed by the USDA-ARS Germplasm Enhancement (GEM) and Truman State University had larger R...

  18. Properties of thermoplastic starch from cassave bagasse and cassava starch and their blends with poly (lactic acid).

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cassava bagasse is an inexpensive and broadly available waste byproduct from cassava starch production. It contains roughly 50% cassava starch along with mostly fiber and could be a valuable feedstock for various bioproducts. Cassava bagasse and cassava starch were used in this study to make fiber-r...

  19. Structural characteristics of slowly digestible starch and resistant starch isolated from heat-moisture treated waxy potato starch.

    PubMed

    Lee, Chang Joo; Moon, Tae Wha

    2015-07-10

    The objective of this study was to investigate the structural characteristics of slowly digestible starch (SDS) and resistant starch (RS) fractions isolated from heat-moisture treated waxy potato starch. The waxy potato starch with 25.7% moisture content was heated at 120°C for 5.3h. Scanning electron micrographs of the cross sections of RS and SDS+RS fractions revealed a growth ring structure. The branch chain-length distribution of debranched amylopectin from the RS fraction had a higher proportion of long chains (DP ≥ 37) than the SDS+RS fraction. The X-ray diffraction intensities of RS and SDS+RS fractions were increased compared to the control. The SDS+RS fraction showed a lower gelatinization enthalpy than the control while the RS fraction had a higher value than the SDS+RS fraction. In this study we showed the RS fraction is composed mainly of crystalline structure and the SDS fraction consists of weak crystallites and amorphous regions. PMID:25857975

  20. Structural characteristics of slowly digestible starch and resistant starch isolated from heat-moisture treated waxy potato starch.

    PubMed

    Lee, Chang Joo; Moon, Tae Wha

    2015-07-10

    The objective of this study was to investigate the structural characteristics of slowly digestible starch (SDS) and resistant starch (RS) fractions isolated from heat-moisture treated waxy potato starch. The waxy potato starch with 25.7% moisture content was heated at 120°C for 5.3h. Scanning electron micrographs of the cross sections of RS and SDS+RS fractions revealed a growth ring structure. The branch chain-length distribution of debranched amylopectin from the RS fraction had a higher proportion of long chains (DP ≥ 37) than the SDS+RS fraction. The X-ray diffraction intensities of RS and SDS+RS fractions were increased compared to the control. The SDS+RS fraction showed a lower gelatinization enthalpy than the control while the RS fraction had a higher value than the SDS+RS fraction. In this study we showed the RS fraction is composed mainly of crystalline structure and the SDS fraction consists of weak crystallites and amorphous regions.

  1. Mutations in PHKA2 are responsible for X-linked liver glycogen storage disease

    SciTech Connect

    Hendrickx, J.; Coucke, P.; Dams, E.

    1994-09-01

    X-linked liver glycogenosis type I (XLG I) is due to a deficiency of phosphorylase kinase (PHK), a key enzyme in the control of glycogen breakdown. XLG I is the most common glycogen storage disease. Patients show hepatomegaly, growth retardation and elevation of liver enzymes as their main clinical symptoms. We assigned the XLG I gene to the chromosomal region Xp22 by linkage analysis in six XLG I families. As the liver {alpha}-subunit of PHK (PHKA2) was also localized to Xp22, PHKA2 was considered a candidate gene for XLG I. In this study, we searched for mutations in 6 exons of the PHDA2 gene of 9 unrelated XLG I patients by SSCP analysis. This revealed three point mutations present in three different patients. Two of these mutations introduce a premature stop codon leading to a truncated protein. The third mutation abolishes a 5{prime} splice site consensus sequence leading to exon skipping. All three mutations therefore result in a PHKA2 protein that lacks several amino acids, what most probably affects enzyme function or stability. These findings indicate that PHKA2 is the XLG I gene.

  2. Development and Characterization of Spaghetti with High Resistant Starch Content Supplemented with Banana Starch

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Pasta products, such as spaghetti, are relatively healthy foods traditionally manufactured from durum wheat semolina and water. Nutritionally improved spaghetti products with additional health benefits can be produced by supplementing durum wheat with suitable food additives, such as banana starch....

  3. A suite of Lotus japonicus starch mutants reveals both conserved and novel features of starch metabolism.

    PubMed

    Vriet, Cécile; Welham, Tracey; Brachmann, Andreas; Pike, Marilyn; Pike, Jodie; Perry, Jillian; Parniske, Martin; Sato, Shusei; Tabata, Satoshi; Smith, Alison M; Wang, Trevor L

    2010-10-01

    The metabolism of starch is of central importance for many aspects of plant growth and development. Information on leaf starch metabolism other than in Arabidopsis (Arabidopsis thaliana) is scarce. Furthermore, its importance in several agronomically important traits exemplified by legumes remains to be investigated. To address this issue, we have provided detailed information on the genes involved in starch metabolism in Lotus japonicus and have characterized a comprehensive collection of forward and TILLING (for Targeting Induced Local Lesions IN Genomes) reverse genetics mutants affecting five enzymes of starch synthesis and two enzymes of starch degradation. The mutants provide new insights into the structure-function relationships of ADP-glucose pyrophosphorylase and glucan, water dikinase1 in particular. Analyses of the mutant phenotypes indicate that the pathways of leaf starch metabolism in L. japonicus and Arabidopsis are largely conserved. However, the importance of these pathways for plant growth and development differs substantially between the two species. Whereas essentially starchless Arabidopsis plants lacking plastidial phosphoglucomutase grow slowly relative to wild-type plants, the equivalent mutant of L. japonicus grows normally even in a 12-h photoperiod. In contrast, the loss of GLUCAN, WATER DIKINASE1, required for starch degradation, has a far greater effect on plant growth and fertility in L. japonicus than in Arabidopsis. Moreover, we have also identified several mutants likely to be affected in new components or regulators of the pathways of starch metabolism. This suite of mutants provides a substantial new resource for further investigations of the partitioning of carbon and its importance for symbiotic nitrogen fixation, legume seed development, and perenniality and vegetative regrowth.

  4. Resistant Starch: Promise for Improving Human Health12

    PubMed Central

    Birt, Diane F.; Boylston, Terri; Hendrich, Suzanne; Jane, Jay-Lin; Hollis, James; Li, Li; McClelland, John; Moore, Samuel; Phillips, Gregory J.; Rowling, Matthew; Schalinske, Kevin; Scott, M. Paul; Whitley, Elizabeth M.

    2013-01-01

    Ongoing research to develop digestion-resistant starch for human health promotion integrates the disciplines of starch chemistry, agronomy, analytical chemistry, food science, nutrition, pathology, and microbiology. The objectives of this research include identifying components of starch structure that confer digestion resistance, developing novel plants and starches, and modifying foods to incorporate these starches. Furthermore, recent and ongoing studies address the impact of digestion-resistant starches on the prevention and control of chronic human diseases, including diabetes, colon cancer, and obesity. This review provides a transdisciplinary overview of this field, including a description of types of resistant starches; factors in plants that affect digestion resistance; methods for starch analysis; challenges in developing food products with resistant starches; mammalian intestinal and gut bacterial metabolism; potential effects on gut microbiota; and impacts and mechanisms for the prevention and control of colon cancer, diabetes, and obesity. Although this has been an active area of research and considerable progress has been made, many questions regarding how to best use digestion-resistant starches in human diets for disease prevention must be answered before the full potential of resistant starches can be realized. PMID:24228189

  5. Evaluation of a High Throughput Starch Analysis Optimised for Wood

    PubMed Central

    Bellasio, Chandra; Fini, Alessio; Ferrini, Francesco

    2014-01-01

    Starch is the most important long-term reserve in trees, and the analysis of starch is therefore useful source of physiological information. Currently published protocols for wood starch analysis impose several limitations, such as long procedures and a neutralization step. The high-throughput standard protocols for starch analysis in food and feed represent a valuable alternative. However, they have not been optimised or tested with woody samples. These have particular chemical and structural characteristics, including the presence of interfering secondary metabolites, low reactivity of starch, and low starch content. In this study, a standard method for starch analysis used for food and feed (AOAC standard method 996.11) was optimised to improve precision and accuracy for the analysis of starch in wood. Key modifications were introduced in the digestion conditions and in the glucose assay. The optimised protocol was then evaluated through 430 starch analyses of standards at known starch content, matrix polysaccharides, and wood collected from three organs (roots, twigs, mature wood) of four species (coniferous and flowering plants). The optimised protocol proved to be remarkably precise and accurate (3%), suitable for a high throughput routine analysis (35 samples a day) of specimens with a starch content between 40 mg and 21 µg. Samples may include lignified organs of coniferous and flowering plants and non-lignified organs, such as leaves, fruits and rhizomes. PMID:24523863

  6. [Inheritance analysis of resistant starch content in kernels of wheat].

    PubMed

    Pang, Huan; Li, Wei-Hua; Zhang, Hong-Bin; Wang, Lin; Yin, Yong-An; Yuan, Hui-Gong; Wang, Zi-Bu

    2010-02-01

    In this study, three wheat (Triticum aestivum L.) cultivars with high and low levels of resistant starch contents each were selected to obtain 15 F1 combinations from a diallel cross without reciprocals to be used to study the inheritance of resistant starch content. The results of this study are useful to select new wheat cultivar with high level of resistant starch content. Annong 90202 and D68-20 were the best among the wheat cultivars tested for general combining ability of resistant starch content, which significantly increased the resistant starch content in its progenies. The specific combining ability of Annong 90202 x 04 Dan 28 and 06-5 x D68-20 were the best among the F1 combinations, and the values of specific combining ability effects were significantly higher than other combinations. The inheritance of resistant starch content fitted the additive-dominance model, and the degree of dominance was super dominance. The alleles for increasing resistant starch content were recessive. The distribution of alleles for increasing and reducing resistant starch contents in the parental lines was not even. The number of recessive alleles for resistant starch content was greater than the dominant alleles. Annong 90202 and 04 Dan 28 had more recessive genes controlling resistant starch content, while Ningchun 18 and Xinchun 5 had more dominant genes. The narrow sense heritability of resistant starch content was 36.49%.

  7. Slowly digestible starch: concept, mechanism, and proposed extended glycemic index.

    PubMed

    Zhang, Genyi; Hamaker, Bruce R

    2009-11-01

    Starch is the major glycemic carbohydrate in foods, and its nutritional property is related to its rate and extent of digestion and absorption in the small intestine. A classification of starch into rapidly digestible starch (RDS), slowly digestible starch (SDS), and resistant starch (RS) based on the in vitro Englyst test is used to specify the nutritional quality of starch. Both the RDS and RS fractions have been extensively studied while there are only limited studies on the intermediate starch fraction of SDS, particularly regarding its structural basis and slow digestion mechanism. The current understanding of SDS including its concept, measurement method, structural basis and mechanism, physiological consequences, and approaches to make SDS is reviewed. An in vivo method of extended glycemic index (EGI) is proposed to evaluate its metabolic effect and related health consequences.

  8. Drying and cracking mechanisms in a starch slurry.

    PubMed

    Goehring, Lucas

    2009-09-01

    Starch-water slurries are commonly used to study fracture dynamics. Drying starch cakes benefit from being simple, economical, and reproducible systems, and have been used to model desiccation fracture in soils, thin-film fracture in paint, and columnar joints in lava. In this paper, the physical properties of starch-water mixtures are studied, and used to interpret and develop a multiphase transport model of drying. Starch cakes are observed to have a nonlinear elastic modulus, and a desiccation strain that is comparable to that generated by their maximum achievable capillary pressure. It is shown that a large material porosity is divided between pore spaces between starch grains, and pores within starch grains. This division of pore space leads to two distinct drying regimes, controlled by liquid and vapor transport of water, respectively. The relatively unique ability for drying starch to generate columnar fracture patterns is shown to be linked to the unusually strong separation of these two transport mechanisms.

  9. Preparation, physicochemical characterization and application of acetylated lotus rhizome starches.

    PubMed

    Sun, Suling; Zhang, Ganwei; Ma, Chaoyang

    2016-01-01

    Acetylated lotus rhizome starches were prepared, physicochemically characterized and used as food additives in puddings. The percentage content of the acetyl groups and degree of substitution increased linearly with the amount of acetic anhydride used. The introduction of acetyl groups was confirmed via Fourier transform infrared (FT-IR) spectroscopy. The values of the pasting parameters were lower for acetylated starch than for native starch. Acetylation was found to increase the light transmittance (%), the freeze-thaw stability, the swelling power and the solubility of the starch. Sensorial scores for puddings prepared using native and acetylated lotus rhizome starches as food additives indicated that puddings produced from the modified starches with superior properties over those prepared from native starch. PMID:26453845

  10. Properties of a glycogen like polysaccharide produced by a mutant of Escherichia coli lacking glycogen synthase and maltodextrin phosphorylase.

    PubMed

    Kwak, Ji-Yun; Kim, Min-Gyu; Kim, Young-Wan; Ban, Hyun-Seung; Won, Mi-Sun; Park, Jong-Tae; Park, Kwan-Hwa

    2016-01-20

    Escherichia coli mutant TBP38 lacks glycogen synthase (GlgA) and maltodextrin phosphorylase (MalP). When grown on maltose in fed-batch fermentation TBP38 accumulated more than 50-fold higher glycogen-type polysaccharide than its parental strain. The polysaccharides were extracted at different growth stages and migrated as one peak in size-exclusion chromatography. TBP38 produced polysaccharides ranging 2.6 × 10(6)-4.6 × 10(6)Da. A ratio of short side-chains (DP ≦ 12) in the polysaccharides was greater than 50%, and number-average degree of polymerization varied from 9.8 to 8.4. The polysaccharides showed 70-290 times greater water-solubility than amylopectin. Km values using porcine and human pancreatic α-amylases with polysaccharides were 2- to 4-fold larger than that of amylopectin. kcat values were similar for both α-amylases. The TBP38 polysaccharides had 40-60% lower digestibility to amyloglucosidase than amylopectin. Intriguingly, the polysaccharides showed strong immunostimulating effects on mouse macrophage cell comparable to lipopolysaccharides. The lipopolysaccharide contamination levels were too low to account for this effect. PMID:26572397

  11. RNase E forms a complex with polynucleotide phosphorylase in cyanobacteria via a cyanobacterial-specific nonapeptide in the noncatalytic region.

    PubMed

    Zhang, Ju-Yuan; Deng, Xue-Mei; Li, Feng-Pu; Wang, Li; Huang, Qiao-Yun; Zhang, Cheng-Cai; Chen, Wen-Li

    2014-04-01

    RNase E, a central component involved in bacterial RNA metabolism, usually has a highly conserved N-terminal catalytic domain but an extremely divergent C-terminal domain. While the C-terminal domain of RNase E in Escherichia coli recruits other components to form an RNA degradation complex, it is unknown if a similar function can be found for RNase E in other organisms due to the divergent feature of this domain. Here, we provide evidence showing that RNase E forms a complex with another essential ribonuclease-the polynucleotide phosphorylase (PNPase)-in cyanobacteria, a group of ecologically important and phylogenetically ancient organisms. Sequence alignment for all cyanobacterial RNase E proteins revealed several conserved and variable subregions in their noncatalytic domains. One such subregion, an extremely conserved nonapeptide (RRRRRRSSA) located near the very end of RNase E, serves as the PNPase recognition site in both the filamentous cyanobacterium Anabaena PCC7120 and the unicellular cyanobacterium Synechocystis PCC6803. These results indicate that RNase E and PNPase form a ribonuclease complex via a common mechanism in cyanobacteria. The PNPase-recognition motif in cyanobacterial RNase E is distinct from those previously identified in Proteobacteria, implying a mechanism of coevolution for PNPase and RNase E in different organisms.

  12. Crystal structure of Caulobacter crescentus polynucleotide phosphorylase reveals a mechanism of RNA substrate channelling and RNA degradosome assembly.

    PubMed

    Hardwick, Steven W; Gubbey, Tobias; Hug, Isabelle; Jenal, Urs; Luisi, Ben F

    2012-04-01

    Polynucleotide phosphorylase (PNPase) is an exoribonuclease that cleaves single-stranded RNA substrates with 3'-5' directionality and processive behaviour. Its ring-like, trimeric architecture creates a central channel where phosphorolytic active sites reside. One face of the ring is decorated with RNA-binding K-homology (KH) and S1 domains, but exactly how these domains help to direct the 3' end of single-stranded RNA substrates towards the active sites is an unsolved puzzle. Insight into this process is provided by our crystal structures of RNA-bound and apo Caulobacter crescentus PNPase. In the RNA-free form, the S1 domains adopt a 'splayed' conformation that may facilitate capture of RNA substrates. In the RNA-bound structure, the three KH domains collectively close upon the RNA and direct the 3' end towards a constricted aperture at the entrance of the central channel. The KH domains make non-equivalent interactions with the RNA, and there is a marked asymmetry within the catalytic core of the enzyme. On the basis of these data, we propose that structural non-equivalence, induced upon RNA binding, helps to channel substrate to the active sites through mechanical ratcheting. Structural and biochemical analyses also reveal the basis for PNPase association with RNase E in the multi-enzyme RNA degradosome assembly of the α-proteobacteria.

  13. Transition state analogue inhibitors of human methylthioadenosine phosphorylase and bacterial methylthioadenosine/S-adenosylhomocysteine nucleosidase incorporating acyclic ribooxacarbenium ion mimics

    PubMed Central

    Clinch, Keith; Evans, Gary B.; Fröhlich, Richard F. G.; Gulab, Shivali A.; Gutierrez, Jemy A.; Mason, Jennifer M.; Schramm, Vern L.; Tyler, Peter C.; Woolhouse, Anthony D.

    2012-01-01

    Several acyclic hydroxy-methylthio-amines with 3 to 5 carbon atoms were prepared and coupled via a methylene link to 9-deazaadenine. The products were tested for inhibition against human MTAP and E. coli and N. meningitidis MTANs and gave Ki values as low as 0.23 nM. These results were compared to those obtained with 1st and 2nd generation inhibitors (1S)-1-(9-deazaadenin-9-yl)-1,4-dideoxy-1,4-imino-5-methylthio-d-ribitol (MT-Immucillin-A, 3) and (3R,4S)-1-[9-deazaadenin-9-yl)methyl]3-hydroxy-4-methylthiomethylpyrrolidine (MT-DADMe-Immucillin-A, 4). The best inhibitors were found to exhibit binding affinities of approximately 2- to 4-fold those of 3 but were significantly weaker than 4. Cleavage of the 2,3 carbon–carbon bond in MT-Immucillin-A (3) gave an acyclic product (79) with a 21,500 fold loss of activity against E. coli MTAN. In another case, N-methylation of a side chain secondary amine resulted in a 250-fold loss of activity against the same enzyme [(±)-65 vs (±)-68]. The inhibition results were also contrasted with those acyclic derivatives previously prepared as inhibitors for a related enzyme, purine nucleoside phosphorylase (PNP), where some inhibitors in the latter case were found to be more potent than their cyclic counterparts. PMID:22854195

  14. Development of a capillary electrophoresis method for analyzing adenosine deaminase and purine nucleoside phosphorylase and its application in inhibitor screening.

    PubMed

    Qi, Yanfei; Li, Youxin; Bao, James J

    2016-08-01

    A novel capillary electrophoresis (CE) method was developed for simultaneous analysis of adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) in red blood cells (RBCs). The developed method considered and took advantage of the natural conversion from the ADA product, inosine to hypoxanthine. The transformation ratio was introduced for ADA and PNP analysis to obtain more reliable results. After optimizing the enzymatic incubation and electrophoresis separation conditions, the determined activities of ADA and PNP in 12 human RBCs were 0.237-0.833 U/ml and 9.013-10.453 U/ml packed cells, respectively. The analysis of ADA in mice RBCs indicated that there was an apparent activity difference between healthy and hepatoma mice. In addition, the proposed method was also successfully applied in the inhibitor screening from nine traditional Chinese medicines, and data showed that ADA activities were strongly inhibited by Rhizoma Chuanxiong and Angelica sinensis. The inhibition effect of Angelica sinensis on ADA is first reported here and could also inhibit PNP activity. PMID:27173606

  15. Isotope-specific and amino acid-specific heavy atom substitutions alter barrier crossing in human purine nucleoside phosphorylase.

    PubMed

    Suarez, Javier; Schramm, Vern L

    2015-09-01

    Computational chemistry predicts that atomic motions on the femtosecond timescale are coupled to transition-state formation (barrier-crossing) in human purine nucleoside phosphorylase (PNP). The prediction is experimentally supported by slowed catalytic site chemistry in isotopically labeled PNP (13C, 15N, and 2H). However, other explanations are possible, including altered volume or bond polarization from carbon-deuterium bonds or propagation of the femtosecond bond motions into slower (nanoseconds to milliseconds) motions of the larger protein architecture to alter catalytic site chemistry. We address these possibilities by analysis of chemistry rates in isotope-specific labeled PNPs. Catalytic site chemistry was slowed for both [2H]PNP and [13C, 15N]PNP in proportion to their altered protein masses. Secondary effects emanating from carbon-deuterium bond properties can therefore be eliminated. Heavy-enzyme mass effects were probed for local or global contributions to catalytic site chemistry by generating [15N, 2H]His8-PNP. Of the eight His per subunit, three participate in contacts to the bound reactants and five are remote from the catalytic sites. [15N, 2H]His8-PNP had reduced catalytic site chemistry larger than proportional to the enzymatic mass difference. Altered barrier crossing when only His are heavy supports local catalytic site femtosecond perturbations coupled to transition-state formation. Isotope-specific and amino acid specific labels extend the use of heavy enzyme methods to distinguish global from local isotope effects.

  16. Isotope-specific and amino acid-specific heavy atom substitutions alter barrier crossing in human purine nucleoside phosphorylase.

    PubMed

    Suarez, Javier; Schramm, Vern L

    2015-09-01

    Computational chemistry predicts that atomic motions on the femtosecond timescale are coupled to transition-state formation (barrier-crossing) in human purine nucleoside phosphorylase (PNP). The prediction is experimentally supported by slowed catalytic site chemistry in isotopically labeled PNP (13C, 15N, and 2H). However, other explanations are possible, including altered volume or bond polarization from carbon-deuterium bonds or propagation of the femtosecond bond motions into slower (nanoseconds to milliseconds) motions of the larger protein architecture to alter catalytic site chemistry. We address these possibilities by analysis of chemistry rates in isotope-specific labeled PNPs. Catalytic site chemistry was slowed for both [2H]PNP and [13C, 15N]PNP in proportion to their altered protein masses. Secondary effects emanating from carbon-deuterium bond properties can therefore be eliminated. Heavy-enzyme mass effects were probed for local or global contributions to catalytic site chemistry by generating [15N, 2H]His8-PNP. Of the eight His per subunit, three participate in contacts to the bound reactants and five are remote from the catalytic sites. [15N, 2H]His8-PNP had reduced catalytic site chemistry larger than proportional to the enzymatic mass difference. Altered barrier crossing when only His are heavy supports local catalytic site femtosecond perturbations coupled to transition-state formation. Isotope-specific and amino acid specific labels extend the use of heavy enzyme methods to distinguish global from local isotope effects. PMID:26305965

  17. Polynucleotide Phosphorylase Regulates Multiple Virulence Factors and the Stabilities of Small RNAs RsmY/Z in Pseudomonas aeruginosa

    PubMed Central

    Chen, Ronghao; Weng, Yuding; Zhu, Feng; Jin, Yongxin; Liu, Chang; Pan, Xiaolei; Xia, Bin; Cheng, Zhihui; Jin, Shouguang; Wu, Weihui

    2016-01-01

    Post-transcriptional regulation enables bacteria to quickly response to environmental stresses. Polynucleotide phosphorylase (PNPase), which contains an N-terminal catalytic core and C-terminal RNA binding KH-S1 domains, is involved in RNA processing. Here we demonstrate that in Pseudomonas aeruginosa the KH-S1 domains of PNPase are required for the type III secretion system (T3SS) and bacterial virulence. Transcriptome analysis revealed a pleiotropic role of PNPase in gene regulation. Particularly, the RNA level of exsA was decreased in the ΔKH-S1 mutant, which was responsible for the reduced T3SS expression. Meanwhile, the pilus biosynthesis genes were down regulated and the type VI secretion system (T6SS) genes were up regulated in the ΔKH-S1 mutant, which were caused by increased levels of small RNAs, RsmY, and RsmZ. Further studies revealed that deletion of the KH-S1 domains did not affect the transcription of RsmY/Z, but increased their stabilities. An in vivo pull-down and in vitro electrophoretic mobility shift assay (EMSA) demonstrated a direct interaction between RsmY/Z and the KH-S1 fragment. Overall, this study reveals the roles of PNPase in the regulation of virulence factors and stabilities of small RNAs in P. aeruginosa. PMID:26973625

  18. Purine nucleoside phosphorylase and xanthine oxidase activities in erythrocytes and plasma from marine, semiaquatic and terrestrial mammals.

    PubMed

    López-Cruz, Roberto I; Pérez-Milicua, Myrna Barjau; Crocker, Daniel E; Gaxiola-Robles, Ramón; Bernal-Vertiz, Jaime A; de la Rosa, Alejandro; Vázquez-Medina, José P; Zenteno-Savín, Tania

    2014-05-01

    Purine nucleoside phosphorylase (PNP) and xanthine oxidase (XO) are key enzymes involved in the purine salvage pathway. PNP metabolizes purine bases to synthetize purine nucleotides whereas XO catalyzes the oxidation of purines to uric acid. In humans, PNP activity is reported to be high in erythrocytes and XO activity to be low in plasma; however, XO activity increases after ischemic events. XO activity in plasma of northern elephant seals has been reported during prolonged fasting and rest and voluntary associated apneas. The objective of this study was to analyze circulating PNP and XO activities in marine mammals adapted to tolerate repeated cycles of ischemia/reperfusion associated with diving (bottlenose dolphin, northern elephant seal) in comparison with semiaquatic (river otter) and terrestrial mammals (human, pig). PNP activities in plasma and erythrocytes, as well as XO activity in plasma, from all species were quantified by spectrophotometry. No clear relationship in circulating PNP or XO activity could be established between marine, semiaquatic and terrestrial mammals. Erythrocytes from bottlenose dolphins and humans are highly permeable to nucleosides and glucose, intraerythrocyte PNP activity may be related to a release of purine nucleotides from the liver. High-energy costs will probably mean a higher ATP degradation rate in river otters, as compared to northern elephant seals or dolphins. Lower erythrocyte PNP activity and elevated plasma XO activity in northern elephant seal could be associated with fasting and/or sleep- and dive-associated apneas.

  19. Purine nucleoside phosphorylase and the enzymatic antioxidant defense system in breast milk from women with different levels of arsenic exposure.

    PubMed

    Gaxiola-Robles, Ramón; Labrada-Martagón, Vanessa; Bitzer-Quintero, Oscar Kurt; Zenteno-Savín, Tania; Méndez-Rodríguez, Lía Celina

    2015-05-01

    Purine nucleoside phosphorylase (PNP) is an ubiquitous enzyme which plays an important role in arsenic (As) detoxification. As is a toxic metalloid present in air, soil and water; is abundant in the environment and is readily transferred along the trophic chain, being found even in human breast milk. Milk is the main nutrient source for the growth and development of neonates. Information on breast milk synthesis and its potential defense mechanism against As toxicity is scarce. In this study, PNP and antioxidant enzymes activities, as well as glutathione (GSH) and total arsenic (TAs) concentrations, were quantified in breast milk samples. PNP, superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), glutathione peroxidase (GPx), glutathione reductase (GR) activities and GSH concentration were determined spectrophotometrically; TAs concentration ([TAs]) was measured by atomic absorption spectrometry. Data suggest an increase in PNP activity (median = 0.034 U mg protein-1) in the presence of TAs (median = 1.16 g L(-1)). To explain the possible association of PNP activity in breast milk with the activity of the antioxidant enzymes as well as with GSH and TAs concentrations, generalized linear models were built. In the adjusted model, GPx and GR activities showed a statistically significant (p<0.01) association with PNP activity. These results may suggest that PNP activity increases in the presence of TAs as part of the detoxification mechanism in breast milk.

  20. A study on the interaction of rhodamine B with methylthioadenosine phosphorylase protein sourced from an Antarctic soil metagenomic library.

    PubMed

    Bartasun, Paulina; Cieśliński, Hubert; Bujacz, Anna; Wierzbicka-Woś, Anna; Kur, Józef

    2013-01-01

    The presented study examines the phenomenon of the fluorescence under UV light excitation (312 nm) of E. coli cells expressing a novel metagenomic-derived putative methylthioadenosine phosphorylase gene, called rsfp, grown on LB agar supplemented with a fluorescent dye rhodamine B. For this purpose, an rsfp gene was cloned and expressed in an LMG194 E. coli strain using an arabinose promoter. The resulting RSFP protein was purified and its UV-VIS absorbance spectrum and emission spectrum were assayed. Simultaneously, the same spectroscopic studies were carried out for rhodamine B in the absence or presence of RSFP protein or native E. coli proteins, respectively. The results of the spectroscopic studies suggested that the fluorescence of E. coli cells expressing rsfp gene under UV illumination is due to the interaction of rhodamine B molecules with the RSFP protein. Finally, this interaction was proved by a crystallographic study and then by site-directed mutagenesis of rsfp gene sequence. The crystal structures of RSFP apo form (1.98 Å) and complex RSFP/RB (1.90 Å) show a trimer of RSFP molecules located on the crystallographic six fold screw axis. The RSFP complex with rhodamine B revealed the binding site for RB, in the pocket located on the interface between symmetry related monomers.

  1. Activity of glycogen synthase and glycogen phosphorylase in normal and cirrhotic rat liver during glycogen synthesis from glucose or fructose.

    PubMed

    Bezborodkina, Natalia N; Chestnova, Anna Yu; Okovity, Sergey V; Kudryavtsev, Boris N

    2014-03-01

    Cirrhotic patients often demonstrate glucose intolerance, one of the possible causes being a decreased glycogen-synthesizing capacity of the liver. At the same time, information about the rates of glycogen synthesis in the cirrhotic liver is scanty and contradictory. We studied the dynamics of glycogen accumulation and the activity of glycogen synthase (GS) and glycogen phosphorylase (GP) in the course of 120min after per os administration of glucose or fructose to fasted rats with CCl4-cirrhosis or fasted normal rats. Blood serum and liver pieces were sampled for examinations. In the normal rat liver administration of glucose/fructose initiated a fast accumulation of glycogen, while in the cirrhotic liver glycogen was accumulated with a 20min delay and at a lower rate. In the normal liver GS activity rose sharply and GPa activity dropped in the beginning of glycogen synthesis, but 60min later a high synthesis rate was sustained at the background of a high GS and GPa activity. Contrariwise, in the cirrhotic liver glycogen was accumulated at the background of a decreased GS activity and a low GPa activity. Refeeding with fructose resulted in a faster increase in the GS activity in both the normal and the cirrhotic liver than refeeding with glucose. To conclude, the rate of glycogen synthesis in the cirrhotic liver is lower than in the normal one, the difference being probably associated with a low GS activity.

  2. Design of vectors for efficient expression of human purine nucleoside phosphorylase in skin fibroblasts from enzyme-deficient humans

    SciTech Connect

    Osborne, W.R.A.; Miller, A.D.

    1988-09-01

    Purine nucleoside phosphorylase deficiency is an inherited disorder associated with a severe immune defect that is fatal. Enzyme replacement therapy is an attractive approach to treatment of this disease. To this aim the authors constructed retroviral vectors containing a human PNP cDNA and a selectable gene encoding neomycin phosphotransferase. PNP expression was controlled by either the early promoter from simian virus 40, the immediate early promoter from human cytomegalovirus, or the retroviral promoter. Cultured skin fibroblasts from two unrelated PNP-deficient patients that were infected with these vectors expressed mean PNP activities of 0.03, 0.74, and 5.9 /mu/mol/hr per mg of protein, respectively. The latter infectants had PNP activities eight times the level of 0.74 /mu/mol/hr per mg of protein observed in normal skin fibroblasts, enabling rapid metabolism of exogenous deoxyguanosine, the cytotoxic metabolite that accumulates in the plasma of PNP-deficient patients. These experiments indicate that viral long terminal repeat was the strongest promoter for expression of PNP and suggest the potential of human skin fibroblasts as vehicles for therapeutic gene expression.

  3. Characterization of the Methylthioadenosine Phosphorylase Polymorphism rs7023954 - Incidence and Effects on Enzymatic Function in Malignant Melanoma

    PubMed Central

    Limm, Katharina; Dettmer, Katja; Reinders, Jörg; Oefner, Peter J.; Bosserhoff, Anja-Katrin

    2016-01-01

    Deficiency of methylthioadenosine phosphorylase (MTAP) supports melanoma development and progression through accumulation of its substrate 5’-methylthioadenosine (MTA), which leads amongst others to a constitutive inhibition of protein arginine methyltransferases (PRMTs) and activation of the transcription factor AP-1 via the receptor ADORA2B. Genetic association studies have also suggested that genetic polymorphism in MTAP may modulate the risk of melanoma. Here, we investigated the only globally common non-synonymous single nucleotide polymorphism (SNP) reported to date for MTAP. The SNP rs7023954 is located in exon 3 (c.166G>A), and leads to the conservative substitution of one branched-chain amino acid residue (valine) for another (isoleucine) at position 56 (p.Val56Ile). Whereas genotype frequencies in normal and primary melanoma tissues or cell lines were in Hardy-Weinberg equilibrium based on cDNA amplicon sequencing, a marked (P = 0.00019) deviation was observed in metastatic melanoma tissues and cell lines due to a deficit of heterozygotes. Enzyme assays conducted on the co-dominantly expressed alleles revealed no difference in the conversion rate of MTA to adenine and 5-methylthioribose-1-phosphate, indicating that this known enzymatic activity does not modulate the tumor suppressive function of MTAP. PMID:27479139

  4. The kinetic mechanism of human uridine phosphorylase 1: Towards the development of enzyme inhibitors for cancer chemotherapy.

    PubMed

    Renck, Daiana; Ducati, Rodrigo G; Palma, Mario S; Santos, Diógenes S; Basso, Luiz A

    2010-05-01

    Uridine phosphorylase (UP) is a key enzyme in the pyrimidine salvage pathway, catalyzing the reversible phosphorolysis of uridine to uracil and ribose-1-phosphate (R1P). The human UP type 1 (hUP1) is a molecular target for the design of inhibitors intended to boost endogenous uridine levels to rescue normal tissues from the toxicity of fluoropyrimidine nucleoside chemotherapeutic agents, such as capecitabine and 5-fluorouracil. Here, we describe a method to obtain homogeneous recombinant hUP1, and present initial velocity, product inhibition, and equilibrium binding data. These results suggest that hUP1 catalyzes uridine phosphorolysis by a steady-state ordered bi bi kinetic mechanism, in which inorganic phosphate binds first followed by the binding of uridine, and uracil dissociates first, followed by R1P release. Fluorescence titration at equilibrium showed cooperative binding of either P(i) or R1P binding to hUP1. Amino acid residues involved in either catalysis or substrate binding were proposed based on pH-rate profiles.

  5. Purine nucleoside phosphorylase and xanthine oxidase activities in erythrocytes and plasma from marine, semiaquatic and terrestrial mammals.

    PubMed

    López-Cruz, Roberto I; Pérez-Milicua, Myrna Barjau; Crocker, Daniel E; Gaxiola-Robles, Ramón; Bernal-Vertiz, Jaime A; de la Rosa, Alejandro; Vázquez-Medina, José P; Zenteno-Savín, Tania

    2014-05-01

    Purine nucleoside phosphorylase (PNP) and xanthine oxidase (XO) are key enzymes involved in the purine salvage pathway. PNP metabolizes purine bases to synthetize purine nucleotides whereas XO catalyzes the oxidation of purines to uric acid. In humans, PNP activity is reported to be high in erythrocytes and XO activity to be low in plasma; however, XO activity increases after ischemic events. XO activity in plasma of northern elephant seals has been reported during prolonged fasting and rest and voluntary associated apneas. The objective of this study was to analyze circulating PNP and XO activities in marine mammals adapted to tolerate repeated cycles of ischemia/reperfusion associated with diving (bottlenose dolphin, northern elephant seal) in comparison with semiaquatic (river otter) and terrestrial mammals (human, pig). PNP activities in plasma and erythrocytes, as well as XO activity in plasma, from all species were quantified by spectrophotometry. No clear relationship in circulating PNP or XO activity could be established between marine, semiaquatic and terrestrial mammals. Erythrocytes from bottlenose dolphins and humans are highly permeable to nucleosides and glucose, intraerythrocyte PNP activity may be related to a release of purine nucleotides from the liver. High-energy costs will probably mean a higher ATP degradation rate in river otters, as compared to northern elephant seals or dolphins. Lower erythrocyte PNP activity and elevated plasma XO activity in northern elephant seal could be associated with fasting and/or sleep- and dive-associated apneas. PMID:24530799

  6. Isotope-specific and amino acid-specific heavy atom substitutions alter barrier crossing in human purine nucleoside phosphorylase

    PubMed Central

    Suarez, Javier; Schramm, Vern L.

    2015-01-01

    Computational chemistry predicts that atomic motions on the femtosecond timescale are coupled to transition-state formation (barrier-crossing) in human purine nucleoside phosphorylase (PNP). The prediction is experimentally supported by slowed catalytic site chemistry in isotopically labeled PNP (13C, 15N, and 2H). However, other explanations are possible, including altered volume or bond polarization from carbon-deuterium bonds or propagation of the femtosecond bond motions into slower (nanoseconds to milliseconds) motions of the larger protein architecture to alter catalytic site chemistry. We address these possibilities by analysis of chemistry rates in isotope-specific labeled PNPs. Catalytic site chemistry was slowed for both [2H]PNP and [13C, 15N]PNP in proportion to their altered protein masses. Secondary effects emanating from carbon–deuterium bond properties can therefore be eliminated. Heavy-enzyme mass effects were probed for local or global contributions to catalytic site chemistry by generating [15N, 2H]His8-PNP. Of the eight His per subunit, three participate in contacts to the bound reactants and five are remote from the catalytic sites. [15N, 2H]His8-PNP had reduced catalytic site chemistry larger than proportional to the enzymatic mass difference. Altered barrier crossing when only His are heavy supports local catalytic site femtosecond perturbations coupled to transition-state formation. Isotope-specific and amino acid specific labels extend the use of heavy enzyme methods to distinguish global from local isotope effects. PMID:26305965

  7. A Ca(2+)-dependent global conformational change in the 3D structure of phosphorylase kinase obtained from electron microscopy.

    PubMed

    Nadeau, Owen W; Carlson, Gerald M; Gogol, Edward P

    2002-01-01

    Phosphorylase kinase (PhK), a Ca(2+)-dependent regulatory enzyme of the glycogenolytic cascade in skeletal muscle, is a 1.3 MDa hexadecameric oligomer comprising four copies of four distinct subunits, termed alpha, beta, gamma, and delta, the last being endogenous calmodulin. The structures of both nonactivated and Ca(2+)-activated PhK were determined to elucidate Ca(2+)-induced structural changes associated with PhK's activation. Reconstructions of both conformers of the kinase, each including over 11,000 particles, yielded bridged, bilobal structures with resolutions estimated by Fourier shell correlation at 24 A using a 0.5 correlation cutoff, or at 18 A by the 3sigma (corrected for D(2) symmetry) threshold curve. Extensive Ca(2+)-induced structural changes were observed in regions encompassing both the lobes and bridges, consistent with changes in subunit interactions upon activation. The relative placement of the alpha, beta, gamma, and delta subunits in the nonactivated three-dimensional structure, relying upon previous two-dimensional localizations, is in agreement with the known effects of Ca(2+) on subunit conformations and interactions in the PhK complex. PMID:11796107

  8. Increasing free-energy (ATP) conservation in maltose-grown Saccharomyces cerevisiae by expression of a heterologous maltose phosphorylase.

    PubMed

    de Kok, Stefan; Yilmaz, Duygu; Suir, Erwin; Pronk, Jack T; Daran, Jean-Marc; van Maris, Antonius J A

    2011-09-01

    Increasing free-energy conservation from the conversion of substrate into product is crucial for further development of many biotechnological processes. In theory, replacing the hydrolysis of disaccharides by a phosphorolytic cleavage reaction provides an opportunity to increase the ATP yield on the disaccharide. To test this concept, we first deleted the native maltose metabolism genes in Saccharomyces cerevisiae. The knockout strain showed no maltose-transport activity and a very low residual maltase activity (0.03 μmol mg protein(-1)min(-1)). Expression of a maltose phosphorylase gene from Lactobacillus sanfranciscensis and the MAL11 maltose-transporter gene resulted in relatively slow growth (μ(aerobic) 0.09 ± 0.03 h(-1)). Co-expression of Lactococcus lactis β-phosphoglucomutase accelerated maltose utilization via this route (μ(aerobic) 0.21 ± 0.01 h(-1), μ(anaerobic) 0.10 ± 0.00 h(-1)). Replacing maltose hydrolysis with phosphorolysis increased the anaerobic biomass yield on maltose in anaerobic maltose-limited chemostat cultures by 26%, thus demonstrating the potential of phosphorolysis to improve the free-energy conservation of disaccharide metabolism in industrial microorganisms.

  9. Analysis of octenylsuccinate rice and tapioca starches: Distribution of octenylsuccinic anhydride groups in starch granules.

    PubMed

    Whitney, Kristin; Reuhs, Bradley L; Ovando Martinez, Maribel; Simsek, Senay

    2016-11-15

    Characterization of the fine structure of octenylsuccinic anhydride (OSA) starch would lead to a better understanding of functional properties. OSA rice and tapioca starches were analyzed using microscopy, liquid chromatography and nuclear magnetic resonance. Chain length distribution of amylopectin changed significantly (P<0.05) after OSA esterification. Weight averaged degree of polymerization (DPw) decreased significantly (P<0.05) from 16.47 to 13.29 and from 14.87 to 12.47 in native and OSA rice and tapioca starches, respectively. The chain length distribution of pure amylopectin fractions suggested that OSA groups were not present in the amylopectin portion of the starch. (1)H NMR analysis of pure amylose and amylopectin fractions indicated that OSA substitution was present only in amylose fractions of rice and tapioca starches. Esterification with 3% OSA results in starch that has OSA substituted mainly on amylose chains or possibly on amylopectin chains that have been hydrolyzed from the amylopectin molecules during esterification. PMID:27283674

  10. Novel products from starch based feedstocks

    Technology Transfer Automated Retrieval System (TEKTRAN)

    There has been progress in the utilization of starch as a partial replacement for petroleum based plastics, but it remains a poor direct substitute for plastics, and a moderate one for composites. Our research focuses on using polymers produced from direct fermentation such as poly(lactic acid) or m...

  11. Heat expanded starch-based compositions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A heat expansion process similar to that used for expanded bead polystyrene was used to expand starch-based compositions. Foam beads made by solvent extraction had the appearance of polystyrene beads but their open-cell structure precluded them from expanding further when heated. Non-porous beads, p...

  12. Starch: chemistry, microstructure, processing and enzymatic degradation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Starch is recognized as one of the most abundant and important commodities containing value added attributes for a vast number of industrial applications. Its chemistry, structure, property and susceptibility to various chemical, physical and enzymatic modifications offer a high technological value ...

  13. Iodine catalyzed acetylation of starch and cellulose

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Starch and cellulose, earth's most abundant biopolymers, are of tremendous economic importance. Over 90% of cotton and 50% of wood are made of cellulose. Wood and cotton are the major resources for all cellulose products such as paper, textiles, construction materials, cardboard, as well as such c...

  14. Resistant starch in food: a review.

    PubMed

    Raigond, Pinky; Ezekiel, Rajarathnam; Raigond, Baswaraj

    2015-08-15

    The nutritional property of starch is related to its rate and extent of digestion and absorption in the small intestine. For nutritional purposes, starch is classified as rapidly available, slowly available and resistant starch (RS). The exact underlying mechanism of relative resistance of starch granules is complicated because those factors are often interconnected. The content of RS in food is highly influenced by food preparation manner and processing techniques. Physical or chemical treatments also alter the level of RS in a food. Commercial preparations of RS are now available and can be added to foods as an ingredient for lowering the calorific value and improving textural and organoleptic characteristics along with increasing the amount of dietary fiber. RS has assumed great importance owing to its unique functional properties and health benefits. The beneficial effects of RS include glycemic control and control of fasting plasma triglyceride and cholesterol levels and absorption of minerals. This review attempts to analyze the information published, especially in the recent past, on classification, structure, properties, applications and health benefits of RS.

  15. Reactions of Starch in Ionic Liquids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We found that starches are found to be soluble at 80 ºC in ionic liquids such as 1-butyl-3-methylimidazolium chloride (BMIMCl) and 1-butyl-3-methylimidazolium dicyanamide (BMIMdca) in concentration up to 10% (w/w). Higher concentrations of biopolymers in these novel solvents resulted in solutions w...

  16. Insight into the redox regulation of the phosphoglucan phosphatase SEX4 involved in starch degradation.

    PubMed

    Silver, Dylan M; Silva, Leslie P; Issakidis-Bourguet, Emmanuelle; Glaring, Mikkel A; Schriemer, David C; Moorhead, Greg B G

    2013-01-01

    Starch is the major carbohydrate reserve in plants, and is degraded for growth at night. Starch breakdown requires reversible glucan phosphorylation at the granule surface by novel dikinases and phosphatases. The dual-specificity phosphatase starch excess 4 (SEX4) is required for glucan desphosphorylation; however, regulation of the enzymatic activity of SEX4 is not well understood. We show that SEX4 switches between reduced (active) and oxidized (inactive) states, suggesting that SEX4 is redox-regulated. Although only partial reactivation of SEX4 was achieved using artificial reductants (e.g. dithiothreitol), use of numerous chloroplastic thioredoxins recovered activity completely, suggesting that thioredoxins could reduce SEX4 in vivo. Analysis of peptides from oxidized and reduced SEX4 identified a disulfide linkage between the catalytic cysteine at position 198 (Cys198) and the cysteine at position 130 (Cys130) within the phosphatase domain. The position of these cysteines was structurally analogous to that for known redox-regulated dual-specificity phosphatases, suggesting a common mechanism of reversible oxidation amongst these phosphatases. Mutation of Cys130 renders SEX4 more sensitive to oxidative inactivation and less responsive to reductive reactivation. Together, these results provide the first biochemical evidence for a redox-dependent structural switch that regulates SEX4 activity, which represents the first plant phosphatase known to undergo reversible oxidation via disulfide bond formation like its mammalian counterparts.

  17. The genomic signature of dog domestication reveals adaptation to a starch-rich diet.

    PubMed

    Axelsson, Erik; Ratnakumar, Abhirami; Arendt, Maja-Louise; Maqbool, Khurram; Webster, Matthew T; Perloski, Michele; Liberg, Olof; Arnemo, Jon M; Hedhammar, Ake; Lindblad-Toh, Kerstin

    2013-03-21

    The domestication of dogs was an important episode in the development of human civilization. The precise timing and location of this event is debated and little is known about the genetic changes that accompanied the transformation of ancient wolves into domestic dogs. Here we conduct whole-genome resequencing of dogs and wolves to identify 3.8 million genetic variants used to identify 36 genomic regions that probably represent targets for selection during dog domestication. Nineteen of these regions contain genes important in brain function, eight of which belong to nervous system development pathways and potentially underlie behavioural changes central to dog domestication. Ten genes with key roles in starch digestion and fat metabolism also show signals of selection. We identify candidate mutations in key genes and provide functional support for an increased starch digestion in dogs relative to wolves. Our results indicate that novel adaptations allowing the early ancestors of modern dogs to thrive on a diet rich in starch, relative to the carnivorous diet of wolves, constituted a crucial step in the early domestication of dogs.

  18. Using the Hexaploid Nature of Wheat To Create Variability in Starch Characteristics.

    PubMed

    Inokuma, Takayuki; Vrinten, Patricia; Shimbata, Tomoya; Sunohara, Ai; Ito, Hiroyuki; Saito, Mika; Taniguchi, Yoshinori; Nakamura, Toshiki

    2016-02-01

    In hexaploid crops, such as bread wheat, it should be possible to fine-tune phenotypic traits by identifying wild-type and null genes from each of the three genomes and combining them in a calculated manner. Here, we demonstrate this with gene combinations for two starch synthesis genes, SSIIa and GBSSI. Lines with inactive copies of both enzymes show a very dramatic change in phenotype, so to create intermediate phenotypes, we used marker-assisted selection to develop near-isogenic lines (NILs) carrying homozygous combinations of null alleles. For both genes, gene dosage effects follow the order B > D ≥ A; therefore, we completed detailed analysis of starch characteristics for NIL 3-3, which is null for the B-genome copy of the SSIIa and GBSSI genes, and NIL 5-5, which has null mutations in the B- and D-genome-encoded copies of both of these genes. The effects of the combinations on phenotypic traits followed the order expected on the basis of genotype, with NIL 5-5 showing the largest differences from the wild type, while NIL 3-3 characteristics were intermediate between NIL 5-5 and the wild type. Differences among genotypes were significant for many starch characteristics, including percent amylose, chain length distribution, gelatinization temperature, retrogradation, and pasting properties, and these differences appeared to translate into improvements in end-product quality, since bread made from type 5-5 flour showed a 3 day lag in staling.

  19. The genomic signature of dog domestication reveals adaptation to a starch-rich diet.

    PubMed

    Axelsson, Erik; Ratnakumar, Abhirami; Arendt, Maja-Louise; Maqbool, Khurram; Webster, Matthew T; Perloski, Michele; Liberg, Olof; Arnemo, Jon M; Hedhammar, Ake; Lindblad-Toh, Kerstin

    2013-03-21

    The domestication of dogs was an important episode in the development of human civilization. The precise timing and location of this event is debated and little is known about the genetic changes that accompanied the transformation of ancient wolves into domestic dogs. Here we conduct whole-genome resequencing of dogs and wolves to identify 3.8 million genetic variants used to identify 36 genomic regions that probably represent targets for selection during dog domestication. Nineteen of these regions contain genes important in brain function, eight of which belong to nervous system development pathways and potentially underlie behavioural changes central to dog domestication. Ten genes with key roles in starch digestion and fat metabolism also show signals of selection. We identify candidate mutations in key genes and provide functional support for an increased starch digestion in dogs relative to wolves. Our results indicate that novel adaptations allowing the early ancestors of modern dogs to thrive on a diet rich in starch, relative to the carnivorous diet of wolves, constituted a crucial step in the early domestication of dogs. PMID:23354050

  20. Continuous enzymatic liquefaction of starch for saccharification

    SciTech Connect

    Carr, M.E.; Black, L.T.; Bagby, M.O.

    1982-01-01

    A process was explored for continuous enzymatic liquefaction of corn starch at high concentration and subsequent saccharification to glucose. The process appears to be quite efficient for conversion of starch to glucose and enzymatic liquefaction and should be readily adaptable to industrial fermentation processes. Preliminary work indicated that milled corn or other cereal grains also can be suitably converted by such a process. Essentially, the process involved incorporation of a thermostable, bacterial alpha-amylase for liquefaction and, subsequently, of a glucoamylase into the continuous mixer under conditions conductive to rapid enzymatic hydrolyses. Also studied was the effect on substrate liquefaction of variables such as starch concentration (40-70%), level of alpha-amylase (0.14-0.4%, dry starch basis), temperature (70-100 degrees C), pH (5.8-7.1), and residence time (6 and 12 minutes). The degree of liquefaction was assessed by determining 1) the Brookfield viscosity, 2) the amount of reducing groups, and 3) the rate and extent of glucose formed after glucoamylase treatment. Best liquefaction processing conditions were achieved by using 50-60% starch concentration, at 95 degrees C, with 0.4% alpha-amylase, and a 6 minute residence period in the mixer. Under these conditions, rates and extents of glucose obtained after glucoamylase treatment approached those obtained in longer laboratory batch liquefactions. The amount of glucose formed in 24 hours with the use of 0.4% glucoamylase was 86% of theory after a 6-min continuous liquefaction, compared to 90% for a 30-min laboratory batch liquefaction (95 degrees C, 0.4% alpha-amylase). (Refs. 15).

  1. Development of oxidised and heat-moisture treated potato starch film.

    PubMed

    Zavareze, Elessandra da Rosa; Pinto, Vânia Zanella; Klein, Bruna; El Halal, Shanise Lisie Mello; Elias, Moacir Cardoso; Prentice-Hernández, Carlos; Dias, Alvaro Renato Guerra

    2012-05-01

    This study investigated the effects of sodium hypochlorite oxidation and a heat-moisture treatment of potato starch on the physicochemical, pasting and textural properties of potato starches in addition to the water vapour permeability (WVP) and mechanical properties of potato starch films produced from these starches. The carbonyl contents, carboxyl contents, swelling power, solubility, pasting properties and gel texture of the native, oxidised and heat-moisture treated (HMT) starches were evaluated. The films made of native, oxidised and HMT starches were characterised by thickness, water solubility, colour, opacity, mechanical properties and WVP. The oxidised and HMT starches had lower viscosity and swelling power compared to the native starch. The films produced from oxidised potato starch had decreased solubility, elongation and WVP values in addition to increased tensile strength compared to the native starch films. The HMT starch increased the tensile strength and WVP of the starch films compared to the native starch. PMID:26434300

  2. Properties of foam and composite materials made o starch and cellulose fiber

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Composite materials were made of starch and cellulose fibers. Pre-gelatinized starch was effective in dispersing pulp fiber in a starch matrix to form a viscous starch/fiber dough. The starch/fiber dough was a useful feedstock for various composite foam and plastic materials. Viscous blends of star...

  3. Production and characterization of cellulose reinforced starch (CRT) films.

    PubMed

    Sudharsan, K; Chandra Mohan, C; Azhagu Saravana Babu, P; Archana, G; Sabina, K; Sivarajan, M; Sukumar, M

    2016-02-01

    Starch from Tamarind seed is considered to be a nonedible and inexpensive component, with many industrial applications. Extraction and characterization of tamarind seed starch was carried out for the synthesis of biopolymer. Tamarind seeds were collected, cleaned and further roasted, decorticated, and pulverized to get starch powder. Total starch content present in each tamarind seed is estimated to be around 65-70%. About 84.68% purified starch can be recovered from the tamarind seed. Defatted Tamarind seed starch has an amylose content of 27.55 wt.% and 72.45 wt.% of amylopectin. Morphological (SEM) and X-ray diffraction were used to evaluate crystallinity. Likewise, TGA and DSC of starch have also been analyzed. Thermal properties of starch obtained from tamarind seeds showed good thermal stability when compared to other starch sources such as Mesquite seed and Mango kernel. This study proved that the tamarind seed starch can be used as a potential biopolymer material. Thermo-stable biofilms were produced through initial optimization studies. Predictive response surface quadratic models were constructed for prediction and optimization of biofilm mechanical properties. Correlation coefficient values were calculated to me more than 0.90 for mechanical responses which implies the fitness of constructed model with experimental data.

  4. Characterization of banana starches obtained from cultivars grown in Brazil.

    PubMed

    de Barros Mesquita, Camila; Leonel, Magali; Franco, Célia Maria Landi; Leonel, Sarita; Garcia, Emerson Loli; Dos Santos, Thaís Paes Rodrigues

    2016-08-01

    The starch market is constantly evolving and studies that provide information about the physical and rheological properties of native starches to meet the diverse demands of the sector are increasingly necessary. In this study starches obtained from five cultivars of banana were analyzed for size and shape of granules, crystallinity, chemical composition, resistant starch, swelling power, solubility, thermal and paste properties. The granules of starch were large (36.58-47.24μm), oval, showed crystallinity pattern type B and the index of crystallinity ranged from 31.94 to 34.06%. The phosphorus content ranged from 0.003 to 0.011%, the amylose ranged from 25.13 to 29.01% and the resistant starch ranged from 65.70 to 80.28%. The starches showed high peak viscosity and breakdown, especially those obtained from 'Nanicão' and 'Grand Naine'. Peak temperature of gelatinization was around 71°C, the enthalpy change (ΔH) ranged from 9.45 to 14.73Jg(-1). The starch from 'Grand Naine' showed higher swelling power (15.19gg(-1)) and the starch from 'Prata-Anã' higher solubility (11.61%). The starches studied are highlighted by their physical and chemical characteristics and may be used in several applications.

  5. Characters related to higher starch accumulation in cassava storage roots

    PubMed Central

    Li, You-Zhi; Zhao, Jian-Yu; Wu, San-Min; Fan, Xian-Wei; Luo, Xing-Lu; Chen, Bao-Shan

    2016-01-01

    Cassava (Manihot esculenta) is valued mainly for high content starch in its roots. Our understanding of mechanisms promoting high starch accumulation in the roots is, however, still very limited. Two field-grown cassava cultivars, Huanan 124(H124) with low root starch and Fuxuan 01(F01) with high root starch, were characterised comparatively at four main growth stages. Changes in key sugars in the leaves, stems and roots seemed not to be strongly associated with the final amount of starch accumulated in the roots. However, when compared with H124, F01 exhibited a more compact arrangement of xylem vascular bundles in the leaf axils, much less callose around the phloem sieve plates in the stems, higher starch synthesis-related enzymatic activity but lower amylase activity in the roots, more significantly up-regulated expression of related genes, and a much higher stem flow rate (SFR). In conclusion, higher starch accumulation in the roots results from the concurrent effects of powerful stem transport capacity highlighted by higher SFR, high starch synthesis but low starch degradation in the roots, and high expression of sugar transporter genes in the stems. A model of high starch accumulation in cassava roots was therefore proposed and discussed. PMID:26892156

  6. Characterization of banana starches obtained from cultivars grown in Brazil.

    PubMed

    de Barros Mesquita, Camila; Leonel, Magali; Franco, Célia Maria Landi; Leonel, Sarita; Garcia, Emerson Loli; Dos Santos, Thaís Paes Rodrigues

    2016-08-01

    The starch market is constantly evolving and studies that provide information about the physical and rheological properties of native starches to meet the diverse demands of the sector are increasingly necessary. In this study starches obtained from five cultivars of banana were analyzed for size and shape of granules, crystallinity, chemical composition, resistant starch, swelling power, solubility, thermal and paste properties. The granules of starch were large (36.58-47.24μm), oval, showed crystallinity pattern type B and the index of crystallinity ranged from 31.94 to 34.06%. The phosphorus content ranged from 0.003 to 0.011%, the amylose ranged from 25.13 to 29.01% and the resistant starch ranged from 65.70 to 80.28%. The starches showed high peak viscosity and breakdown, especially those obtained from 'Nanicão' and 'Grand Naine'. Peak temperature of gelatinization was around 71°C, the enthalpy change (ΔH) ranged from 9.45 to 14.73Jg(-1). The starch from 'Grand Naine' showed higher swelling power (15.19gg(-1)) and the starch from 'Prata-Anã' higher solubility (11.61%). The starches studied are highlighted by their physical and chemical characteristics and may be used in several applications. PMID:27180297

  7. Susceptibility of glutinous rice starch to digestive enzymes.

    PubMed

    Guo, Li; Zhang, Juanjuan; Hu, Jian; Li, Xueling; Du, Xianfeng

    2015-09-01

    To understand the susceptibility of glutinous rice starch to digestive enzymes and its potential impact on glycemic response, enzyme kinetics and in vitro digestibility of the native and gelatinized starches were investigated. The results showed that the Km values of the native and gelatinized starch were 10.35 mg/mL and 9.92 mg/mL, respectively. The digestion rate coefficients k values of the native and gelatinized starches were 2.0 × 10(-3)min(-1) and 1.1 × 10(-2)min(-1), respectively. The contents of rapid digestible starch (RDS), slowly digestible starch (SDS) and resistant starch (RS) in native glutinous rice starch were 8.92%, 21.52% and 69.56%, respectively. After gelatinization, the amounts of RDS, SDS and RS were 18.47%, 29.75% and 51.78%, respectively. The native and gelatinized glutinous rice starches were 10.34% and 14.07% for hydrolysis index (HI), as well as 43.14% and 45.92% for glycemic index (GI), respectively. During the in vitro digestion, the crystallinity of native glutinous rice starch was increased from 34.7% to 35.8% and 38.4% after 20 and 120 min, respectively.

  8. Zinc chloride aqueous solution as a solvent for starch.

    PubMed

    Lin, Meiying; Shang, Xiaoqin; Liu, Peng; Xie, Fengwei; Chen, Xiaodong; Sun, Yongyi; Wan, Junyan

    2016-01-20

    It is important to obtain starch-based homogeneous systems for starch modification. Regarding this, an important key point is to find cheap, low-cost and low-toxicity solvents to allow complete dissolution of starch and its easy regeneration. This study reveals that a ZnCl2 aqueous solution is a good non-derivatizing solvent for starch at 50 °C, and can completely dissolve starch granules. The possible formation of a "zinc-starch complex" might account for the dissolution; and the degradation of starch, which was caused by the H(+) inZnCl2 aqueous solution, could not contribute to full dissolution. From polarized light microscopic observation combined with the solution turbidity results, it was found that the lowest ZnCl2 concentration for full dissolution was 29.6 wt.% at 50 °C, with the dissolving time being 4h. Using Fourier-transform infrared (FTIR), solid state (13)C nuclear magnetic resonance (NMR), and X-ray diffraction (XRD), it was revealed that ZnCl2 solution had no chemical reaction with starch glucosides, but only weakened starch hydrogen bonding and converted the crystalline regions to amorphous regions. In addition, as shown by intrinsic viscosity and thermogravimetric analysis (TGA), ZnCl2 solution caused degradation of starch macromolecules, which was more serious with a higher concentration of ZnCl2 solution.

  9. Characters related to higher starch accumulation in cassava storage roots.

    PubMed

    Li, You-Zhi; Zhao, Jian-Yu; Wu, San-Min; Fan, Xian-Wei; Luo, Xing-Lu; Chen, Bao-Shan

    2016-02-19

    Cassava (Manihot esculenta) is valued mainly for high content starch in its roots. Our understanding of mechanisms promoting high starch accumulation in the roots is, however, still very limited. Two field-grown cassava cultivars, Huanan 124(H124) with low root starch and Fuxuan 01(F01) with high root starch, were characterised comparatively at four main growth stages. Changes in key sugars in the leaves, stems and roots seemed not to be strongly associated with the final amount of starch accumulated in the roots. However, when compared with H124, F01 exhibited a more compact arrangement of xylem vascular bundles in the leaf axils, much less callose around the phloem sieve plates in the stems, higher starch synthesis-related enzymatic activity but lower amylase activity in the roots, more significantly up-regulated expression of related genes, and a much higher stem flow rate (SFR). In conclusion, higher starch accumulation in the roots results from the concurrent effects of powerful stem transport capacity highlighted by higher SFR, high starch synthesis but low starch degradation in the roots, and high expression of sugar transporter genes in the stems. A model of high starch accumulation in cassava roots was therefore proposed and discussed.

  10. Production and characterization of cellulose reinforced starch (CRT) films.

    PubMed

    Sudharsan, K; Chandra Mohan, C; Azhagu Saravana Babu, P; Archana, G; Sabina, K; Sivarajan, M; Sukumar, M

    2016-02-01

    Starch from Tamarind seed is considered to be a nonedible and inexpensive component, with many industrial applications. Extraction and characterization of tamarind seed starch was carried out for the synthesis of biopolymer. Tamarind seeds were collected, cleaned and further roasted, decorticated, and pulverized to get starch powder. Total starch content present in each tamarind seed is estimated to be around 65-70%. About 84.68% purified starch can be recovered from the tamarind seed. Defatted Tamarind seed starch has an amylose content of 27.55 wt.% and 72.45 wt.% of amylopectin. Morphological (SEM) and X-ray diffraction were used to evaluate crystallinity. Likewise, TGA and DSC of starch have also been analyzed. Thermal properties of starch obtained from tamarind seeds showed good thermal stability when compared to other starch sources such as Mesquite seed and Mango kernel. This study proved that the tamarind seed starch can be used as a potential biopolymer material. Thermo-stable biofilms were produced through initial optimization studies. Predictive response surface quadratic models were constructed for prediction and optimization of biofilm mechanical properties. Correlation coefficient values were calculated to me more than 0.90 for mechanical responses which implies the fitness of constructed model with experimental data. PMID:26592701

  11. The potential of resistant starch as a prebiotic.

    PubMed

    Zaman, Siti A; Sarbini, Shahrul R

    2016-01-01

    Resistant starch is defined as the total amount of starch and the products of starch degradation that resists digestion in the small intestine. Starches that were able to resist the digestion will arrive at the colon where they will be fermented by the gut microbiota, producing a variety of products which include short chain fatty acids that can provide a range of physiological benefits. There are several factors that could affect the resistant starch content of a carbohydrate which includes the starch granule morphology, the amylose-amylopectin ratio and its association with other food component. One of the current interests on resistant starch is their potential to be used as a prebiotic, which is a non-digestible food ingredient that benefits the host by stimulating the growth or activity of one or a limited number of beneficial bacteria in the colon. A resistant starch must fulfill three criterions to be classified as a prebiotic; resistance to the upper gastrointestinal environment, fermentation by the intestinal microbiota and selective stimulation of the growth and/or activity of the beneficial bacteria. The market of prebiotic is expected to reach USD 198 million in 2014 led by the export of oligosaccharides. Realizing this, novel carbohydrates such as resistant starch from various starch sources can contribute to the advancement of the prebiotic industry.

  12. Function of pyridoxal 5'-phosphate in glycogen phosphorylase: a model study using 6-fluoro-5'-deoxypyridoxal- and 5'-deoxypyridoxal-reconstituted enzymes

    SciTech Connect

    Chang, Y.C.; Scott, R.D.; Graves, D.J.

    1987-01-27

    A new vitamin B/sub 6/ analogue, 6-fluoro-5'-deoxypyridoxal (6-FDPL), was synthesized and characterized. This analogue, as well as 6-fluoropyridoxal (6-FPAL), 6-fluoropyridoxal phosphate (6-FPLP), and 6-fluoropyridoxine, showed positive heteronuclear /sup 1/H-/sup 18/F nuclear Overhauser effects between the 5'-protons and the 6-fluorine. Apophosphorylase reconstituted with 6-FDLP showed 1% of the activity of the native enzyme in the presence of phosphite. The kinetic pattern, apparent pH optimum of activity, and the activity-temperature dependency of the 6-FDPL-enzyme were virtually identical with those of phosphorylase reconstituted with the parent compound, 6-FPAL except the K/sub m/ of phosphite toward the 6-FDPL-enzyme was 9 times higher than that with the 6-FPAL-enzyme and the 6-FDPL-enzyme showed a lower V/sub max/ value. Phosphorylase reconstituted with 5'-deoxypyridoxal (DPL) also showed activity in the presence of phosphite. The kinetics and the temperature-activity dependency of this reconstituted enzyme were investigated. /sup 19/F nuclear magnetic resonance studies showed that the binding of glucose 1-phosphate to a 6-FDPL-enzyme-adenosine 5'-phosphate (AMP) complex shifted the /sup 19/F signal 0.6 ppm upfield, whereas a 2.1 ppm change was observed when the 6-FPAL-enzyme-AMP formed a complex with glucose 1-phosphate. Analysis of the activation parameters, activation enthalpy and activation entropy, of the reaction of glycogen degradation catalyzed by phosphorylase containing pyridoxal phosphate, 6-FDPL, pyridoxal, or DPL showed that modifications of the coenzyme molecule affected only the activation entropy, not the activation enthalpy. Results of this study indicate that the protein structure surrounding the coenzyme molecule, as well as the coenzyme configuration, is altered upon the binding of ligands.

  13. The GH130 Family of Mannoside Phosphorylases Contains Glycoside Hydrolases That Target β-1,2-Mannosidic Linkages in Candida Mannan*

    PubMed Central

    Cuskin, Fiona; Baslé, Arnaud; Ladevèze, Simon; Day, Alison M.; Gilbert, Harry J.; Davies, Gideon J.; Potocki-Véronèse, Gabrielle; Lowe, Elisabeth C.

    2015-01-01

    The depolymerization of complex glycans is an important biological process that is of considerable interest to environmentally relevant industries. β-Mannose is a major component of plant structural polysaccharides and eukaryotic N-glycans. These linkages are primarily cleaved by glycoside hydrolases, although recently, a family of glycoside phosphorylases, GH130, have also been shown to target β-1,2- and β-1,4-mannosidic linkages. In these phosphorylases, bond cleavage was mediated by a single displacement reaction in which phosphate functions as the catalytic nucleophile. A cohort of GH130 enzymes, however, lack the conserved basic residues that bind the phosphate nucleophile, and it was proposed that these enzymes function as glycoside hydrolases. Here we show that two Bacteroides enzymes, BT3780 and BACOVA_03624, which lack the phosphate binding residues, are indeed β-mannosidases that hydrolyze β-1,2-mannosidic linkages through an inverting mechanism. Because the genes encoding these enzymes are located in genetic loci that orchestrate the depolymerization of yeast α-mannans, it is likely that the two enzymes target the β-1,2-mannose residues that cap the glycan produced by Candida albicans. The crystal structure of BT3780 in complex with mannose bound in the −1 and +1 subsites showed that a pair of glutamates, Glu227 and Glu268, hydrogen bond to O1 of α-mannose, and either of these residues may function as the catalytic base. The candidate catalytic acid and the other residues that interact with the active site mannose are conserved in both GH130 mannoside phosphorylases and β-1,2-mannosidases. Functional phylogeny identified a conserved lysine, Lys199 in BT3780, as a key specificity determinant for β-1,2-mannosidic linkages. PMID:26286752

  14. Reduced enzyme activity and starch level in an induced mutant of chloroplast phosphoglucose isomerase

    SciTech Connect

    Jones, T.W.; Gottlieb, L.D.; Pichersky, E.

    1986-06-01

    Ethyl methane sulfonate treatment was used to induce a mutation in the nuclear gene encoding the chloroplast isozyme of phosphoglucose isomerase in Clarkia xantiana. The mutation, which proved allelic to wild type activity, was backcrossed to wild type for five generations so that the two could be compared in a near isogenic background. An immunological analysis showed that the mutant, when homozygous, reduced the activity of the isozyme by about 50%. In contrast to wild type, the mutant showed little change in leaf starch level over a diurnal period or following a 72-hour continuous light treatment. By the end of the diurnal light period, the mutant accumulated only about 60% as much starch as wild type. However, mutant leaves had an increased sucrose level presumably because photosynthate was directly exported from the chloroplasts. The mutant also exhibited reduced leaf weight. These changes in metabolism and growth suggest that the wild type level of plastid phosphoglucose isomerase activity is necessary to achieve wild type carbohydrate status.

  15. Diadenosine 5',5'''-P1, P4-tetraphosphate alpha, beta-phosphorylase from yeast supports nucleoside diphosphate-phosphate exchange.

    PubMed

    Guranowski, A; Blanquet, S

    1986-05-01

    Homogeneous diadenosine 5',5'''-P1,P4-tetraphosphate alpha, beta-phosphorylase (Ap4A-phosphorylase), the enzyme recently found in yeast (Guranowski, A., and Blanquet, S. (1985) J. Biol. Chem. 260, 3542-3547) catalyzes an exchange reaction between the beta-phosphate of nucleoside diphosphate (NDP) and orthophosphate from the medium (Pi). The common purine and pyrimidine ribonucleoside diphosphates as well as ADP analogs modified either in aglycone, sugar, or at the anhydride bond beta-position are substrates. The Km and rate values for the NDP-Pi exchange reaction were estimated at pH optima. These optima are 6.5 for UDP, 7.0 for ADP or CDP, and 8.0 for GDP. In the presence of 10 mM K2HPO4, 0.1 mM EDTA, and 100 mM Hepes/KOH (pH 7.0), the Km for ADP is 0.7 mM with a rate constant at saturating ADP of 96 s-1. The Km value for orthophosphate is 2 mM. In the NDP-Pi exchange reaction, phosphate can be substituted with arsenate and apparent arsenolysis of NDPs yields corresponding nucleoside monophosphates. The same pH optimum of 6.5 is found for arsenolysis of ADP, GDP, and CDP. Whereas the Ap4A phosphorylase sulfhydryl groups are essential for catalyzing the Ap4A phosphorolysis, the NDP-Pi exchange reactions, and the arsenolysis of NDPs, the divalent metal ions (Mg2+, Mn2+, Ca2+, Co2+, and Cd2+), which had been shown to be essential cofactors of the former reaction, are not required for the two latter ones. Used at concentrations which are optimum for Ap4A phosphorolysis, the cations (particularly Mg2+ and Cd2+) inhibit the NDP-Pi exchange and the arsenolysis of NDPs. Interestingly, the Ap4A phosphorylase exhibits higher specificity for adenosine 5'-phosphosulfate (APS) than for any other NDP tested. The V/Km ratio is almost 5-fold higher with APS than with ADP. However, in the presence of orthophosphate, the APS is irreversibly converted to ADP. Thus, the enzyme displays a property already attributed to ADP-sulfurylase (EC 2.7.7.5), (Grunberg-Manago, M., Del Campillo

  16. Computational Methods for De novo Protein Design and its Applications to the Human Immunodeficiency Virus 1, Purine Nucleoside Phosphorylase, Ubiquitin Specific Protease 7, and Histone Demethylases

    PubMed Central

    Bellows, M.L.; Floudas, C.A.

    2010-01-01

    This paper provides an overview of computational de novo protein design methods, highlighting recent advances and successes. Four protein systems are described that are important targets for drug design: human immunodeficiency virus 1, purine nucleoside phosphorylase, ubiquitin specific protease 7, and histone demethylases. Target areas for drug design for each protein are described, along with known inhibitors, focusing on peptidic inhibitors, but also describing some small-molecule inhibitors. Computational design methods that have been employed in elucidating these inhibitors for each protein are outlined, along with steps that can be taken in order to apply computational protein design to a system that has mainly used experimental methods to date. PMID:20210752

  17. Mixture design of rice flour, maize starch and wheat starch for optimization of gluten free bread quality.

    PubMed

    Mancebo, Camino M; Merino, Cristina; Martínez, Mario M; Gómez, Manuel

    2015-10-01

    Gluten-free bread production requires gluten-free flours or starches. Rice flour and maize starch are two of the most commonly used raw materials. Over recent years, gluten-free wheat starch is available on the market. The aim of this research was to optimize mixtures of rice flour, maize starch and wheat starch using an experimental mixture design. For this purpose, dough rheology and its fermentation behaviour were studied. Quality bread parameters such as specific volume, texture, cell structure, colour and acceptability were also analysed. Generally, starch incorporation reduced G* and increased the bread specific volume and cell density, but the breads obtained were paler than the rice flour breads. Comparing the starches, wheat starch breads had better overall acceptability and had a greater volume than maize-starch bread. The highest value for sensorial acceptability corresponded to the bread produced with a mixture of rice flour (59 g/100 g) and wheat starch (41 g/100 g). PMID:26396377

  18. Cassava starch films containing acetylated starch nanoparticles as reinforcement: Physical and mechanical characterization.

    PubMed

    Teodoro, Ana Paula; Mali, Suzana; Romero, Natália; de Carvalho, Gizilene Maria

    2015-08-01

    This paper reports the use of acetylated starch nanoparticles (NPAac) as reinforcement in thermoplastic starch films. NPAac with an average size of approximately 500 nm were obtained by nanoprecipitation. Fourier transform infrared (FTIR) and thermogravimetric analysis (TGA) indicated that NPAac are more thermally stable and essentially amorphous when compared with acetylated starch. Thermoplastic starch films with different proportions of NPAac (0.5, 1.0, 1.5, 10.0%, w/w) were obtained and characterized by scanning electron microscopy (SEM), water vapor permeability (WVP), adsorption isotherms, TGA and mechanical tests. The inclusion of reinforcement caused changes in film properties: WVP was lowered by 41% for film with 1.5% (w/w) of NPAac and moisture adsorption by 33% for film with 10% (w/w) of NPAac; and the Young's modulus and thermal stability were increased by 162% and 15%, respectively, for film with 0.5% (w/w) of NPAac compared to the starch film without the addition of NPAac.

  19. Mutagenesis of cysteine 81 prevents dimerization of the APS1 subunit of ADP-glucose pyrophosphorylase and alters diurnal starch turnover in Arabidopsis thaliana leaves.

    PubMed

    Hädrich, Nadja; Hendriks, Janneke H M; Kötting, Oliver; Arrivault, Stéphanie; Feil, Regina; Zeeman, Samuel C; Gibon, Yves; Schulze, Waltraud X; Stitt, Mark; Lunn, John E

    2012-04-01

    Many plants, including Arabidopsis thaliana, retain a substantial portion of their photosynthate in leaves in the form of starch, which is remobilized to support metabolism and growth at night. ADP-glucose pyrophosphorylase (AGPase) catalyses the first committed step in the pathway of starch synthesis, the production of ADP-glucose. The enzyme is redox-activated in the light and in response to sucrose accumulation, via reversible breakage of an intermolecular cysteine bridge between the two small (APS1) subunits. The biological function of this regulatory mechanism was investigated by complementing an aps1 null mutant (adg1) with a series of constructs containing a full-length APS1 gene encoding either the wild-type APS1 protein or mutated forms in which one of the five cysteine residues was replaced by serine. Substitution of Cys81 by serine prevented APS1 dimerization, whereas mutation of the other cysteines had no effect. Thus, Cys81 is both necessary and sufficient for dimerization of APS1. Compared to control plants, the adg1/APS1(C81S) lines had higher levels of ADP-glucose and maltose, and either increased rates of starch synthesis or a starch-excess phenotype, depending on the daylength. APS1 protein levels were five- to tenfold lower in adg1/APS1(C81S) lines than in control plants. These results show that redox modulation of AGPase contributes to the diurnal regulation of starch turnover, with inappropriate regulation of the enzyme having an unexpected impact on starch breakdown, and that Cys81 may play an important role in the regulation of AGPase turnover.

  20. A Bacterial Glucanotransferase Can Replace the Complex Maltose Metabolism Required for Starch to Sucrose Conversion in Leaves at Night*

    PubMed Central

    Ruzanski, Christian; Smirnova, Julia; Rejzek, Martin; Cockburn, Darrell; Pedersen, Henriette L.; Pike, Marilyn; Willats, William G. T.; Svensson, Birte; Steup, Martin; Ebenhöh, Oliver; Smith, Alison M.; Field, Robert A.

    2013-01-01

    Controlled conversion of leaf starch to sucrose at night is essential for the normal growth of Arabidopsis. The conversion involves the cytosolic metabolism of maltose to hexose phosphates via an unusual, multidomain protein with 4-glucanotransferase activity, DPE2, believed to transfer glucosyl moieties to a complex heteroglycan prior to their conversion to hexose phosphate via a cytosolic phosphorylase. The significance of this complex pathway is unclear; conversion of maltose to hexose phosphate in bacteria proceeds via a more typical 4-glucanotransferase that does not require a heteroglycan acceptor. It has recently been suggested that DPE2 generates a heterogeneous series of terminal glucan chains on the heteroglycan that acts as a “glucosyl buffer” to ensure a constant rate of sucrose synthesis in the leaf at night. Alternatively, DPE2 and/or the heteroglycan may have specific properties important for their function in the plant. To distinguish between these ideas, we compared the properties of DPE2 with those of the Escherichia coli glucanotransferase MalQ. We found that MalQ cannot use the plant heteroglycan as an acceptor for glucosyl transfer. However, experimental and modeling approaches suggested that it can potentially generate a glucosyl buffer between maltose and hexose phosphate because, unlike DPE2, it can generate polydisperse malto-oligosaccharides from maltose. Consistent with this suggestion, MalQ is capable of restoring an essentially wild-type phenotype when expressed in mutant Arabidopsis plants lacking DPE2. In light of these findings, we discuss the possible evolutionary origins of the complex DPE2-heteroglycan pathway. PMID:23950181

  1. In vitro effect of fenugreek extracts on intestinal sodium-dependent glucose uptake and hepatic glycogen phosphorylase A.

    PubMed

    Al-Habori, M; Raman, A; Lawrence, M J; Skett, P

    2001-01-01

    Fenugreek (Trigonella foenum-graecum L. seed) is a food with traditional medicinal use in diabetes. Beneficial effects have been demonstrated in diabetic animals and both insulin-dependent and non-insulin-dependent diabetic subjects. Effects of a lipid extract A, crude ethanolic extract B, further sub-fractions of B (saponin-free C, saponin D and sapogenin E) and a gum fibre fraction F on intestinal sodium-dependent glucose uptake were investigated in vitro using rabbit intestinal brush border membrane vesicles. All fractions except A inhibited glucose-uptake at 0.33 and/or 3.3 mg/mL (p < 0.001). Greatest inhibition was observed with fractions D and E. Diosgenin and trigonelline (compounds reported in fenugreek) also inhibited glucose-uptake (IC50 values approximately 3 mg/ml, equivalent to 8 mM and 19 mM respectively) but did not account for the activity of the crude extracts. Fenugreek extracts had no effect on basal levels of glycogen phosphorylase a (HGPa) activity in rat hepatocyte suspensions. However fractions C and E caused a marginal but statistically significant inhibition (18.9 and 15.1% respectively, p < 0.05) of glucagon induction of this enzyme suggesting a glucagon-antagonist effect. Diosgenin (1.65 mg/ml; 4 mM) inhibited glucagon-induced HGPa activity by 20% (p < 0.05), and was more effective than trigonelline (non significant inhibition of 9.4% at 1.65 mg/ml, 10 mM). PMID:12369721

  2. The role of glycogen phosphorylase in the regulation of glycogenolysis by insulin and glucagon in isolated eel (Anguilla rostrata) hepatocytes.

    PubMed

    Foster, G D; Moon, T W

    1990-07-01

    The effects of porcine, scombroid, and salmon insulins, and bovine and anglerfish glucagons on glycogen depletion and glycogen phosphorylase (GPase) activities were examined in freshly isolated American eel (Anguilla rostrata) hepatocytes. Eel liver GPase in crude homogenates was activated (increase in % GPase a) by phosphorylating conditions and was rapidly inactivated (less than 1 h) when a phosphatase inhibitor (fluoride) was absent. Caffeine inhibits, and AMP activates, the b form of GPase consistent with their effects on rat liver GPase. Both mammalian and fish glucagons increased glucose production in eel hepatocytes, but had more ambiguous effects on glycogen levels and GPase activities. The magnitude of bovine glucagon effects were dependent on the initial glycogen content of the cells; only at glycogen concentrations less than approximately 70 μmoles.g(-1) did glucagon significantly increase % GPase a. Anglerfish glucagon significantly increased cyclic AMP (cAMP) concentrations by 90% at 10(-7) M, but had no effects at 10(-9) M and 10(-8) M. Scombroid and salmon insulins maintained hepatocyte glycogen concentrations and decreased glucose production, with these effects more pronounced at low (10(-9) to 10(-8) M) rather than high (10(-7) M) hormone concentrations. Porcine and salmon insulins decreased total GPase and % GPase a activities, and salmon insulin decreased CAMP levels, but only at 10(-8) M (by 44%).Glycogen is, therefore, depleted by glucagon and maintained by insulin in freshly isolated American eel hepatocytes, and these changes are accomplished, at least in part, by changes in the activities of GPase. Changes in cAMP do not explain all of the observed hormone effects. PMID:24220919

  3. Preoperative Chemoradiation for Rectal Cancer Using Capecitabine and Celecoxib Correlated With Posttreatment Assessment of Thymidylate Synthase and Thymidine Phosphorylase Expression

    SciTech Connect

    Unger, Keith R.; Romney, Davis A.; Koc, Mehmet; Moskaluk, Christopher A.; Friel, Charles M.; Foley, E.F.; Rich, Tyvin A.

    2011-08-01

    Purpose: Thymidylate synthase (TS) and thymidine phosphorylase (TP) expression have been shown to be predictors of response to therapy. The toxicity, efficacy, surgical morbidity, and immunohistochemical TS and TP expression were assessed in surgical resection specimens after preoperative chemoradiation. Methods and Materials: Twenty patients with clinical stage I to III rectal adenocarcinoma received preoperative chemoradiation and underwent surgical resection 6 weeks later. Results: Posttreatment tumor stages were T1 to T2 and N0 in 30% of patients; T3 to T4 and N0 in 30% of patients; and T1 to T3 and N1 to N2 in 15% of patients. Pathologic complete response (pCR) was evident in 25% and tumor regression occurred in a total of 80% of patients. Anal sphincter-sparing surgery was performed in 80% of cases. Acute and perioperative complications were minimal, with no grade 3/4 toxicity or treatment breaks. Pelvic control was obtained in 90% of patients. With a median follow-up of 65.5 months (range, 8-80 months), the 6-year actuarial survival rate was 75%. Local failure was significantly associated with nonresponse to therapy and with high TS and low TP expression (p = 0.008 and p = 0.04, respectively). Conclusions: The combination of capecitabine, celecoxib, and x-radiation therapy yields excellent response: a 25% pathologic pCR, no acute grade 3/4 toxicity, and minimal surgical morbidity. Nonresponders expressed significantly increased TS levels and decreased TP levels in posttreatment resection specimens compared to responders.

  4. Efficient electrogene therapy for pancreatic adenocarcinoma treatment using the bacterial purine nucleoside phosphorylase suicide gene with fludarabine.

    PubMed

    Deharvengt, Sophie; Rejiba, Soukaina; Wack, Séverine; Aprahamian, Marc; Hajri, Amor

    2007-06-01

    The aim of this study was to demonstrate the potential of electrogene therapy with the bacterial purine nucleoside phosphorylase gene (ePNP), on pancreatic carcinoma (PC) large tumors. The in vivo electroporation (EP) conditions and efficacy were investigated on both subcutaneous xenografts of human PC cells in immunocompromised mice and orthotopic intrapancreatic grafts of rat PC cells in syngenic rats. After intratumoral injection of naked plasmid DNA, EP was performed using a two-needle array with 25-msec pulses and either a 300 V/cm field strength for subcutaneous or a 500 V/cm field strength for orthotopic PC, parameters providing the best electrotransfer as reflected by the measurements of both luciferase activity and ePNP mRNA. As expected, tumors developed sensitivity to prodrug treatment (6-methylpurine deoxyribose or fludarabine phosphate). We observed both significant inhibition of tumor growth and extended survival of treated mice. In fact, after prodrug treatment, PC growth in the subcutaneous model was delayed by 50-70% for ePNP-expressing tumors. In an orthotopic pancreatic tumor model, the animal survival was significantly prolonged after ePNP electrogene transfer followed by fludarabine treatment, with one animal out of 10 being tumor-free 6 months thereafter. The current study demonstrates for the first time on PC the in vivo feasibility of electrogene transfer and its therapeutic efficiency using the suicide gene/prodrug system, ePNP/fludarabine. These findings suggest that electrogene therapy strategy must be considered for pancreatic cancer treatment, particularly at advanced stages of the disease. PMID:17487360

  5. Selective photoregulation of the activity of glycogen synthase and glycogen phosphorylase, two key enzymes in glycogen metabolism.

    PubMed

    Díaz-Lobo, Mireia; Garcia-Amorós, Jaume; Fita, Ignacio; Velasco, Dolores; Guinovart, Joan J; Ferrer, Joan C

    2015-07-14

    Glycogen is a polymer of α-1,4- and α-1,6-linked glucose units that provides a readily available source of energy in living organisms. Glycogen synthase (GS) and glycogen phosphorylase (GP) are the two enzymes that control, respectively, the synthesis and degradation of this polysaccharide and constitute adequate pharmacological targets to modulate cellular glycogen levels, by means of inhibition of their catalytic activity. Here we report on the synthesis and biological evaluation of a selective inhibitor that consists of an azobenzene moiety glycosidically linked to the anomeric carbon of a glucose molecule. In the ground state, the more stable (E)-isomer of the azobenzene glucoside had a slight inhibitory effect on rat muscle GP (RMGP, IC50 = 4.9 mM) and Escherichia coli GS (EcGS, IC50 = 1.6 mM). After irradiation and subsequent conversion to the (Z)-form, the inhibitory potency of the azobenzene glucoside did not significantly change for RMGP (IC50 = 2.4 mM), while its effect on EcGS increased 50-fold (IC50 = 32 μM). Sucrose synthase 4 from potatoes, a glycosyltransferase that does not operate on glycogen, was only slightly inhibited by the (E)-isomer (IC50 = 0.73 mM). These findings could be rationalized on the basis of kinetic and computer-aided docking analysis, which indicated that both isomers of the azobenzene glucoside mimic the EcGS acceptor substrate and exert their inhibitory effect by binding to the glycogen subsite in the active center of the enzyme. The ability to selectively photoregulate the catalytic activity of key enzymes of glycogen metabolism may represent a new approach for the treatment of glycogen metabolism disorders.

  6. Identification of Genes Potentially Regulated by Human Polynucleotide Phosphorylase (hPNPaseold-35) Using Melanoma as a Model

    PubMed Central

    Sokhi, Upneet K.; Bacolod, Manny D.; Dasgupta, Santanu; Emdad, Luni; Das, Swadesh K.; Dumur, Catherine I.; Miles, Michael F.; Sarkar, Devanand; Fisher, Paul B.

    2013-01-01

    Human Polynucleotide Phosphorylase (hPNPaseold-35 or PNPT1) is an evolutionarily conserved 3′→5′ exoribonuclease implicated in the regulation of numerous physiological processes including maintenance of mitochondrial homeostasis, mtRNA import and aging-associated inflammation. From an RNase perspective, little is known about the RNA or miRNA species it targets for degradation or whose expression it regulates; except for c-myc and miR-221. To further elucidate the functional implications of hPNPaseold-35 in cellular physiology, we knocked-down and overexpressed hPNPaseold-35 in human melanoma cells and performed gene expression analyses to identify differentially expressed transcripts. Ingenuity Pathway Analysis indicated that knockdown of hPNPaseold-35 resulted in significant gene expression changes associated with mitochondrial dysfunction and cholesterol biosynthesis; whereas overexpression of hPNPaseold-35 caused global changes in cell-cycle related functions. Additionally, comparative gene expression analyses between our hPNPaseold-35 knockdown and overexpression datasets allowed us to identify 77 potential “direct” and 61 potential “indirect” targets of hPNPaseold-35 which formed correlated networks enriched for cell-cycle and wound healing functional association, respectively. These results provide a comprehensive database of genes responsive to hPNPaseold-35 expression levels; along with the identification new potential candidate genes offering fresh insight into cellular pathways regulated by PNPT1 and which may be used in the future for possible therapeutic intervention in mitochondrial- or inflammation-associated disease phenotypes. PMID:24143183

  7. Ca2+-induced structural changes in phosphorylase kinase detected by small-angle X-ray scattering.

    PubMed

    Priddy, Timothy S; MacDonald, Brian A; Heller, William T; Nadeau, Owen W; Trewhella, Jill; Carlson, Gerald M

    2005-04-01

    Phosphorylase kinase (PhK), a 1.3-MDa (alphabetagammadelta)(4) hexadecameric complex, is a Ca(2+)-dependent regulatory enzyme in the cascade activation of glycogenolysis. PhK comprises two arched (alphabetagammadelta)(2) octameric lobes that are oriented back-to-back with overall D(2) symmetry and joined by connecting bridges. From chemical cross-linking and electron microscopy, it is known that the binding of Ca(2+) by PhK perturbs the structure of all its subunits and promotes redistribution of density throughout both its lobes and bridges; however, little is known concerning the interrelationship of these effects. To measure structural changes induced by Ca(2+) in the PhK complex in solution, small-angle X-ray scattering was performed on nonactivated and Ca(2+)-activated PhK. Although the overall dimensions of the complex were not affected by Ca(2+), the cation did promote a shift in the distribution of the scattering density within the hydrated volume occupied by the PhK molecule, indicating a Ca(2+)-induced conformational change. Computer-generated models, based on elements of the known structure of PhK from electron microscopy, were constructed to aid in the interpretation of the scattering data. Models containing two ellipsoids and four cylinders to represent, respectively, the lobes and bridges of the PhK complex provided theoretical scattering profiles that accurately fit the experimental data. Structural differences between the models representing the nonactivated and Ca(2+)-activated conformers of PhK are consistent with Ca(2+)-induced conformational changes in both the lobes and the interlobal bridges.

  8. Ca2+-induced structural changes in phosphorylase kinase detected by small-angle x-ray scattering

    SciTech Connect

    Priddy, Timothy S.; Macdonald, Brian A.; Heller, William T; Nadeau, Owen W.; Trewhella, Jill; Carlson, Gerald M.

    2005-01-01

    Phosphorylase kinase (PhK), a 1.3-MDa ({alpha}{beta}{gamma}{delta}){sub 4} hexadecameric complex, is a Ca{sup 2+}-dependent regulatory enzyme in the cascade activation of glycogenolysis. PhK comprises two arched ({alpha}{beta}{gamma}{delta}){sub 2} octameric lobes that are oriented back-to-back with overall D{sub 2} symmetry and joined by connecting bridges. From chemical cross-linking and electron microscopy, it is known that the binding of Ca{sup 2+} by PhK perturbs the structure of all its subunits and promotes redistribution of density throughout both its lobes and bridges; however, little is known concerning the interrelationship of these effects. To measure structural changes induced by Ca{sup 2+} in the PhK complex in solution, small-angle X-ray scattering was performed on nonactivated and Ca{sup 2+}-activated PhK. Although the overall dimensions of the complex were not affected by Ca{sup 2+}, the cation did promote a shift in the distribution of the scattering density within the hydrated volume occupied by the PhK molecule, indicating a Ca{sup 2+}-induced conformational change. Computer-generated models, based on elements of the known structure of PhK from electron microscopy, were constructed to aid in the interpretation of the scattering data. Models containing two ellipsoids and four cylinders to represent, respectively, the lobes and bridges of the PhK complex provided theoretical scattering profiles that accurately fit the experimental data. Structural differences between the models representing the nonactivated and Ca{sup 2+}-activated conformers of PhK are consistent with Ca{sup 2+}-induced conformational changes in both the lobes and the interlobal bridges.

  9. Calf spleen purine nucleoside phosphorylase: structure of its ternary complex with an N(7)-acycloguanosine inhibitor and a phosphate anion.

    PubMed

    Luić, M; Koellner, G; Shugar, D; Saenger, W; Bzowska, A

    2001-01-01

    The calf spleen purine nucleoside phosphorylase (PNP) ternary complex with an N(7)-acycloguanosine inhibitor and a phosphate ion has been crystallized in the cubic space group P2(1)3, with unit-cell parameter a = 94.11 A and one monomer per asymmetric unit. X-ray diffraction data were collected using synchrotron radiation (Station X31, EMBL Outstation, DESY, Hamburg). The crystal structure was refined to a resolution of 2.2 A and R and R(free) values of 17.5 and 24.5%, respectively. The acyclonucleoside inhibitor is bound in the active site in an inverted ('upside-down') orientation of the purine base compared with natural substrates. The side chain of Asp243 forms two hydrogen bonds with the base ring: N(delta) donates a hydrogen to N(3) and O(delta) accepts a hydrogen from the guanine N(2)-amino group. N(1)--H of the base is hydrogen bonded to O(epsilon) of Glu201, while N(9) accepts a hydrogen bond from Thr242 O(gamma). In addition, a water molecule (W417) bridges the N(2)-amino group of the base and O(epsilon) of Glu201. In the phosphate-binding site, a phosphate ion is bound to Ser33, His64, Arg84, His86, Ala116 and Ser220. The acyclic chain of the N(7)-acycloguanosine inhibitor is in a folded conformation and together with a water molecule (W388) occupies the pentose-binding site, with possible hydrogen bonds to Tyr88 O(eta) and His257 N(delta 1). This new binding mode fully accounts for the previously observed substrate properties of 7-beta-D-ribofuranosides of hypoxanthine and guanine. It also provides a new starting point for the design of inhibitors of PNP for therapeutic and other applications.

  10. Enzymatic Hydrogen Production from Starch and Water

    SciTech Connect

    Zhang, Y.-H. Percival; Evans, Barbara R; Mielenz, Jonathan R; Hopkins, Robert C.; Adams, Michael W. W.

    2007-01-01

    A novel enzymatic reaction was conducted for producing hydrogen from starch and water at 30oC. The overall reaction comprised of 13 enzymes, 1 cofactor (NADP+), and phosphate was driven by energy stored in carbohydrate starch according to the overall stoichiometry stoichiometric reaction of C6H10O5 (l) + 7 H2O (l) --> 12 H2 (g) + 6 CO2 (g). It is spontaneous and unidirectional because of negative Gibbs free energy and the removal of gaseous products from the aqueous reaction solution. With technology improvement and integration with fuel cells, this technology would be suitable for mobile applications and also solve the challenges associated with hydrogen storage, distribution, and infrastructure in a hydrogen economy.

  11. Prebiotic properties of potato starch dextrins.

    PubMed

    Barczyńska, Renata; Śliżewska, Katarzyna; Libudzisz, Zdzisława; Kapuśniak, Kamila; Kapuśniak, Janusz

    2015-01-01

    The objective of the present study was to compare the prebiotic properties of starch dextrins, that is, resistant dextrins obtained from potato starch in the process of simultaneous thermolysis and chemical modification, which were selected based on previous research. Both prepared dextrins met the definition criterion of dietary fiber and also the basic prebiotic criterion - they were not degraded by the digestive enzymes of the initial sections of the gastrointestinal tract. The growth of probiotic lactobacilli and bifidobacteria, as well as Escherichia coli, Enterococcus, Bacteroides, and Clostridium strains isolated from feces of healthy people, showed that both studied dextrins were utilized as a source of assimilable carbon and energy by the strains. Furthermore, better growth (higher numbers of cells) counts of probiotic bacteria than those of fecal isolates indicated that the studied resistant dextrins showed a selective effect. Both dextrins might be considered as substances with prebiotic properties due to their chemical and physical properties and selectivity towards the studied probiotic bacterial strains.

  12. High throughput screening of starch structures using carbohydrate microarrays.

    PubMed

    Tanackovic, Vanja; Rydahl, Maja Gro; Pedersen, Henriette Lodberg; Motawia, Mohammed Saddik; Shaik, Shahnoor Sultana; Mikkelsen, Maria Dalgaard; Krunic, Susanne Langgaard; Fangel, Jonatan Ulrik; Willats, William George Tycho; Blennow, Andreas

    2016-01-01

    In this study we introduce the starch-recognising carbohydrate binding module family 20 (CBM20) from Aspergillus niger for screening biological variations in starch molecular structure using high throughput carbohydrate microarray technology. Defined linear, branched and phosphorylated maltooligosaccharides, pure starch samples including a variety of different structures with variations in the amylopectin branching pattern, amylose content and phosphate content, enzymatically modified starches and glycogen were included. Using this technique, different important structures, including amylose content and branching degrees could be differentiated in a high throughput fashion. The screening method was validated using transgenic barley grain analysed during development and subjected to germination. Typically, extreme branching or linearity were detected less than normal starch structures. The method offers the potential for rapidly analysing resistant and slowly digested dietary starches. PMID:27468930

  13. High throughput screening of starch structures using carbohydrate microarrays

    PubMed Central

    Tanackovic, Vanja; Rydahl, Maja Gro; Pedersen, Henriette Lodberg; Motawia, Mohammed Saddik; Shaik, Shahnoor Sultana; Mikkelsen, Maria Dalgaard; Krunic, Susanne Langgaard; Fangel, Jonatan Ulrik; Willats, William George Tycho; Blennow, Andreas

    2016-01-01

    In this study we introduce the starch-recognising carbohydrate binding module family 20 (CBM20) from Aspergillus niger for screening biological variations in starch molecular structure using high throughput carbohydrate microarray technology. Defined linear, branched and phosphorylated maltooligosaccharides, pure starch samples including a variety of different structures with variations in the amylopectin branching pattern, amylose content and phosphate content, enzymatically modified starches and glycogen were included. Using this technique, different important structures, including amylose content and branching degrees could be differentiated in a high throughput fashion. The screening method was validated using transgenic barley grain analysed during development and subjected to germination. Typically, extreme branching or linearity were detected less than normal starch structures. The method offers the potential for rapidly analysing resistant and slowly digested dietary starches. PMID:27468930

  14. Synergistic effect of starch on the antibacterial activity of honey.

    PubMed

    Boukraâ, Laïd; Amara, Karim

    2008-03-01

    The role of amylase present in honey in enhancing its antibacterial activity was evaluated in the presence and absence of starch. Two strains of pathogenic bacteria have been used: Staphylococcus aureus and Escherichia coli. For S. aureus, the minimum inhibitory concentration (MIC) for the three varieties of honey tested without starch was 11%, 24%, and 29% (vol/vol), respectively. When starch was added with honey to the media the MIC obtained was 5%, 19%, and 25% (vol/vol), respectively. For E. coli, the MIC for the three varieties without starch was 23%, 28%, and 25% (vol/vol), respectively. When starch was added with honey to media, the MIC was 19%, 26%, and 23% (vol/vol), respectively. It is speculated that the amylase present in honey hydrolyzed the starch chains to randomly produce dextrin and maltose and that this increased the osmotic effect of the media, which consequently increased the antibacterial activity. PMID:18361758

  15. Development of highly-transparent protein/starch-based bioplastics.

    PubMed

    Gonzalez-Gutierrez, J; Partal, P; Garcia-Morales, M; Gallegos, C

    2010-03-01

    Striving to achieve cost-competitive biomass-derived materials for the plastics industry, the incorporation of starch (corn and potato) to a base formulation of albumen and glycerol was considered. To study the effects of formulation and processing, albumen/starch-based bioplastics containing 0-30 wt.% starch were prepared by thermo-plastic and thermo-mechanical processing. Transmittance measurements, DSC, DMTA and tensile tests were performed on the resulting bioplastics. Optical and tensile properties were strongly affected by starch concentration. However, DMTA at low deformation proved to be insensitive to starch addition. Thermo-mechanical processing led to transparent albumen/starch materials with values of strength at low deformation comparable to commodity plastics. Consequently, albumen biopolymers may become a biodegradable alternative to oil-derived plastics for manufacturing transparent packaging and other plastic stuffs.

  16. Anaerobic digestion of cassava starch factory effluent.

    PubMed

    Manilal, V B; Narayanan, C S; Balagopalan, C

    1990-06-01

    Biomethanation of cassava starch factory effluent in a batch digester produced 130 l biogas/kg dry matter with an average melthane content of 59%. About 63% COD was removed during 60 days. In semicontinuous digesters, gas production was 3251/kg dry matter with a retention time of 33,3 days giving a COD reduction of 50%. Size of starter inoculum was important for good biogasification of the effluent.

  17. Effects of citric acid esterification on digestibility, structural and physicochemical properties of cassava starch.

    PubMed

    Mei, Ji-Qiang; Zhou, Da-Nian; Jin, Zheng-Yu; Xu, Xue-Ming; Chen, Han-Qing

    2015-11-15

    In this study, citric acid was used to react with cassava starch in order to compare the digestibility, structural and physicochemical properties of citrate starch samples. The results indicated that citric acid esterification treatment significantly increased the content of resistant starch (RS) in starch samples. The swelling power and solubility of citrate starch samples were lower than those of native starch. Compared with native starch, a new peak at 1724 cm(-1) was appeared in all citrate starch samples, and crystalline peaks of all starch citrates became much smaller or even disappeared. Differential scanning calorimetry results indicated that the endothermic peak of citrate starches gradually shrank or even disappeared. Moreover, the citrate starch gels exhibited better freeze-thaw stability. These results suggested that citric acid esterification induced structural changes in cassava starch significantly affected its digestibility and it could be a potential method for the preparation of RS with thermal stability.

  18. Effect of acetylation, oxidation and annealing on physicochemical properties of bean starch.

    PubMed

    Simsek, Senay; Ovando-Martínez, Maribel; Whitney, Kristin; Bello-Pérez, Luis A

    2012-10-15

    Black and Pinto bean starches were physically and chemically modified to investigate the effect of modification on digestibility and physicochemical properties of bean starch. The impact of acetylation, oxidation (ozonation) and annealing on the chemical composition, syneresis, swelling volume, pasting, thermal properties and digestibility of starches was evaluated. The physicochemical and estimated glycemic index (eGI) of the Black and Pinto bean starches treated with ozone were not significantly (P>0.05) different than that of their respective control starches. Annealed starches had improved thermal and pasting properties compared to native starches. Acetylated starches presented reduced syneresis, good pasting properties and lower eGI. Also, all modified starches had increased levels of resistant starch (RS). Therefore, the digestibility and physicochemical properties of bean starch were affected by the type of modification but there were no significant (P>0.05) differences between the Black and Pinto bean starches.

  19. Effect of dry heating with ionic gums on physicochemical properties of starch.

    PubMed

    Sun, Qingjie; Si, Fumei; Xiong, Liu; Chu, Lijun

    2013-02-15

    Corn starch, potato starch, pea starch were impregnated with ionic gums (sodium alginate, CMC, and xanthan, 1% based on starch solids) and heat-treated in a dry state for 0, 2, or 4 h at 130°C. Effects of the dry heating on paste viscosity (RVA), microstructure and thermal properties were examined. Dry heat treatment with ionic gums reduced the pasting temperature of the three starches. Heating with xanthan increased the paste viscosity of corn and potato starch. With heat treatment, the paste viscosity of all the starch-sodium alginate mixtures decreased. Heating with CMC increased the paste viscosity of potato starch, but decreased that of corn and pea starch. After dry-heating, To, Tp and Tc of potato starch with ionic gums decreased significantly. SEM of potato starch with CMC showed that the gel structure got compacter after drying-heating. Heat treatment obviously improved the functional properties of the three starches. PMID:23194543

  20. Recent advances of starch-based excipients used in extended-release tablets: a review.

    PubMed

    Hong, Yan; Liu, Guodong; Gu, Zhengbiao

    2016-01-01

    In recent years, polysaccharides, including starch and its derivatives, have been widely used in the pharmaceutical industry, including as diluents, fillers, binders, disintegrants and glidants. The use of native starch as excipient in extended-release tablets is limited due to its low compactibility and enzymatic degradability, leading to the formation of weakly structured tablets. To overcome these limitations and expand the application of starch as an excipient, researchers have modified starch by physical and chemical methods, as well as by enzymatic hydrolysis. Some starch derivatives, including retrograded starch, pregelatinized starch, carboxymethyl starch, starch acetate, cross-linked starch and grafted starch have recently been introduced as excipients in oral tablets to control drug release. In this review, applications of starch and its derivatives as extended release excipients are reviewed and future frontiers are described.

  1. Starch composites reinforced by bamboo cellulosic crystals.

    PubMed

    Liu, Dagang; Zhong, Tuhua; Chang, Peter R; Li, Kaifu; Wu, Qinglin

    2010-04-01

    Using a method of combined HNO(3)-KClO(3) treatment and sulfuric acid hydrolysis, bamboo cellulose crystals (BCCs) were prepared and used to reinforce glycerol plasticized starch. The structure and morphology of BCCs were investigated using X-ray diffraction, electron microscopy, and solid-state (13)C NMR. Results showed that BCCs were of typical cellulose I structure, and the morphology was dependent on its concentration in the suspension. BCC of 50-100 nm were assembled into leaf nervations at low concentration (i.e. 0.1 wt.% of solids), but congregated into a micro-sized "flower" geometry at high concentration (i.e. 10.0 wt.% of solids). Tensile strength and Young's modulus of the starch/BCC composite films (SBC) were enhanced by the incorporation of the crystals due to reinforcement of BCCs and reduction of water uptake. BCCs at the optimal 8% loading level exhibited a higher reinforcing efficiency for plasticized starch plastic than any other loading level.

  2. Structure characterization and hypoglycemic effects of dual modified resistant starch from indica rice starch.

    PubMed

    Zhou, Ying; Meng, Shaohua; Chen, Deyi; Zhu, Xiping; Yuan, Huaibo

    2014-03-15

    Hypoglycemic effects of indica rice resistant starch (IR-RS) were investigated. We prepared IR-RS using a method that combined physical modification and enzyme modification, and the RS content was 47.0%. Differential scanning calorimetry--thermal gravimetric analysis showed that IR-RS have higher enthalpy and less loss of mass than single modified RS, heat-moisture RS and native starch. Scanning electron microscopy revealed that IR-RS displayed more compact spatial structure. IR-RS products displayed a mixture of B-and V-type x-ray diffraction patterns and the cyrstallinity was 51.0%. IR-RS significantly affected body weight, blood glucose, organ indices and serum lipid levels. These results demonstrated that dual modification changed the structure of indica rice starch and affected its digestibility as well as the blood glucose levels of the diabetic mice who consumed it.

  3. Thermal and rheological properties of nixtamalized maize starch.

    PubMed

    Mendez-Montealvo, G; Sánchez-Rivera, M M; Paredes-López, O; Bello-Pérez, L A

    2006-12-15

    The effect of nixtamalization process on thermal and rheological characteristics of corn starch was studied. Starch of raw sample had higher gelatinization temperature than its raw counterpart, because, the Ca(2+) ions stabilize starch structure of nixtamalized sample; however, the enthalpy values were not different in both samples. The temperature of the phase transition of the retrograded starches (raw and nixtamalized) were not different at the storage times assessed, but the enthalpy values of the above mentioned transition was different, indicating a lower reorganization of the starch structure in the nixtamalized sample. The viscoamylographic profile showed differences between both starches, since raw starch had higher peak viscosity than the nixtamalized sample due to partial gelatinization of some granules during this heat treatment. Rheological test showed that at low temperature (25 degrees C) the raw and nixtamalized starches presented different behaviour; however, the elastic characteristic was more important in the starch gel structure. The nixtamalization process produced changes in thermal and rheological characteristics becoming important in those products elaborated from nixtamalized maize.

  4. Characterization and development mechanism of Apios americana tuber starch.

    PubMed

    Yangcheng, Hanyu; Belamkar, Vikas; Cannon, Steven B; Jane, Jay-Lin

    2016-10-20

    Apios americana is a wild legume-bearing plant with edible tubers. Domestication of Apios is in progress because of the superior nutritional value and health benefits of the tuber. Objectives of this study were to: (1) characterize physicochemical properties of the Apios tuber starch; and (2) understand differences in starch structures and properties between the mother (seed) and child (progeny) tubers and the mechanism of starch development. Granules of the Apios tuber starch displayed ellipsoidal, rod, or kidney shape with diameter ranges of 1-30μm. The mother tuber starches displayed greater percentage crystallinity, larger gelatinization enthalpy-changes, longer branch-chain lengths of amylopectin, and lower pasting viscosity than their counterpart child tuber starches. The mother tuber starch of Apios 2127 displayed distinct two peaks of gelatinization, which were attributed to starch granules located at different regions of the tuber having different structures and properties. The mother tuber displayed more active starch biosynthesis in the periphery than in the center of the tuber.

  5. Characterization and development mechanism of Apios americana tuber starch.

    PubMed

    Yangcheng, Hanyu; Belamkar, Vikas; Cannon, Steven B; Jane, Jay-Lin

    2016-10-20

    Apios americana is a wild legume-bearing plant with edible tubers. Domestication of Apios is in progress because of the superior nutritional value and health benefits of the tuber. Objectives of this study were to: (1) characterize physicochemical properties of the Apios tuber starch; and (2) understand differences in starch structures and properties between the mother (seed) and child (progeny) tubers and the mechanism of starch development. Granules of the Apios tuber starch displayed ellipsoidal, rod, or kidney shape with diameter ranges of 1-30μm. The mother tuber starches displayed greater percentage crystallinity, larger gelatinization enthalpy-changes, longer branch-chain lengths of amylopectin, and lower pasting viscosity than their counterpart child tuber starches. The mother tuber starch of Apios 2127 displayed distinct two peaks of gelatinization, which were attributed to starch granules located at different regions of the tuber having different structures and properties. The mother tuber displayed more active starch biosynthesis in the periphery than in the center of the tuber. PMID:27474558

  6. Available starch pools in mature soybean leaves. [Glycine max

    SciTech Connect

    Highsmith, M.

    1987-04-01

    Soybeans, Glycine max, were grown in the greenhouse and in the field under conditions that varied CO/sub 2/ levels, irradiance, and photosynthetic period. Photosynthesis, sucrose export rate, starch accumulation, and starch mobilization were measured in mature leaves and compared to expansion of developing leaves two nodes above. Measurements were made daily over a twelve day period. Measured parameters in mature leaves varied daily with night expansion of the growing leaves. Starch accumulation and photosynthesis were affected by the environmental factors while sucrose export rate and starch mobilization were affected only if growth of the expanding leaves changed. The data indicated that influence of night expansion of developing leaves and influence of the environmental factors on photosynthesis caused two pools of starch to be formed in mature leaves. Concentration of each starch pool was calculated. The pool associated with night growth of expanding leaves was positively correlated with photosynthesis and sucrose export rate. The other pool, a storage, was negatively correlated with these parameters. Starch mobilization was comprised of starch from either pool, depending on the plant's environment or it was comprised of starch from both pools.

  7. Pysicochemical properties of Tibetan hull-less barley starch.

    PubMed

    Yangcheng, Hanyu; Gong, Lingxiao; Zhang, Ying; Jane, Jay-lin

    2016-02-10

    Objectives of this study were to (1) determine the starch physicochemical properties of two commercial Tibetan hull-less barley varieties, Beiqing (BQ) and Kangqing (KQ); and (2) understand the relationship between unique properties of the starches, their structures, and impacts of growing conditions. The BQ barleys were grown at a location with lower temperature and less rainfall compared with the KQ barleys. The BQ starches showed significantly lower onset-gelatinization temperature (54.1-54.9 °C), larger gelatinization-temperature range (9.4-10.6 °C), and higher peak-viscosities (138.9-153.9RVU) than the KQ starches (55.1-56.1 °C, 7.4-8.8 °C, and 63.4-64.7RVU, respectively). After a treatment with 2% sodium-dodecyl-sulphate solution, the KQ starches showed substantially greater increases in peak viscosities than the BQ starches. Annealing of starch and enhanced amylose-lipid complex formation, resulting from higher growing temperature during the development of the KQ starches, likely contributed to the differences in thermal and pasting properties between the BQ and KQ starches.

  8. Agro-industrial residue from starch extraction of Pachyrhizus ahipa as filler of thermoplastic corn starch films.

    PubMed

    López, O V; Versino, F; Villar, M A; García, M A

    2015-12-10

    Biocomposites films based on thermoplastic corn starch (TPS) containing 0.5% w/w fibrous residue from Pachyrhizus ahipa starch extraction (PASR) were obtained by melt-mixing and compression molding. PASR is mainly constituted by remaining cell walls and natural fibers, revealed by Scanning Electron Microscopy (SEM). Chemical composition of the residue indicated that fiber and starch were the principal components. Biocomposites thermo-stability was determined by Thermo-Gravimetric Analysis. A continuous PASR-TPS interface was observed by SEM, as a result of a good adhesion of the fibrous residue to starch matrix. Likewise, films containing PASR presented fewer superficial cracks than TPS ones, whereas their fracture surfaces were more irregular. Besides, the presence of PASR increased starch films roughness, due to fibers agglomerates. Films reinforced with PASR showed significantly lower water vapor permeability (WVP). In addition, PARS filler increased maximum tensile strength and Young's modulus of TPS films, thus leading to more resistant starch matrixes. PMID:26428131

  9. Agro-industrial residue from starch extraction of Pachyrhizus ahipa as filler of thermoplastic corn starch films.

    PubMed

    López, O V; Versino, F; Villar, M A; García, M A

    2015-12-10

    Biocomposites films based on thermoplastic corn starch (TPS) containing 0.5% w/w fibrous residue from Pachyrhizus ahipa starch extraction (PASR) were obtained by melt-mixing and compression molding. PASR is mainly constituted by remaining cell walls and natural fibers, revealed by Scanning Electron Microscopy (SEM). Chemical composition of the residue indicated that fiber and starch were the principal components. Biocomposites thermo-stability was determined by Thermo-Gravimetric Analysis. A continuous PASR-TPS interface was observed by SEM, as a result of a good adhesion of the fibrous residue to starch matrix. Likewise, films containing PASR presented fewer superficial cracks than TPS ones, whereas their fracture surfaces were more irregular. Besides, the presence of PASR increased starch films roughness, due to fibers agglomerates. Films reinforced with PASR showed significantly lower water vapor permeability (WVP). In addition, PARS filler increased maximum tensile strength and Young's modulus of TPS films, thus leading to more resistant starch matrixes.

  10. Processing effects on susceptibility of starch to digestion in some dietary starch sources.

    PubMed

    Niba, Lorraine L

    2003-01-01

    Maize flour, potato flour, cocoyam flour, plantain flour, yam flour, and rice flour were assayed for starch digestibility by an established enzymatic procedure. These were either autoclaved, microwaved, or parboiled and then freeze-dried. Freeze-dried samples were stored for 10 days either below freezing or at ambient temperature. Parameters assessed were readily digestible starch (RDS), slowly digestible starch (SDS), and total starch (TS). Data was analyzed by t-test (P < or = 0.05). RDS levels among raw flours ranged from 1.01 g/100g in rice flour to 8.16 g/100 g in cocoyam flour. Autoclaving and parboiling increased RDS levels in most flours, while microwaving significantly reduced RDS compared with raw flour. Ambient temperature storage reduced the RDS content. SDS levels ranged from 4.95 g/100 g in yam flour to 22.2 g/100 g in maize flour. SDS levels were increased by autoclaving and parboling, but significantly reduced by microwaving, compared with the raw flour. Storage at ambient temperature resulted in lower SDS. The TS content in raw flour ranged from 28.0 g/100 g in plantain flour to 68.4 g/100 g in rice flour. Autoclaving resulted in reduced TS levels insome flours. Moist heat processing and the post-process storage temperature therefore result in significant changes in starch susceptibility to enzymic digestion. This information will be useful in developing food processing and storage procedures that modify starch resistance to digestion in order to optimize its nutritional quality and to enhance the physiological benefits.

  11. Hydroxyethyl starch - the importance of being earnest.

    PubMed

    Chappell, Daniel; Jacob, Matthias

    2013-01-01

    Despite ongoing controversial expert discussions the European Medicines Agency (EMA) recently recommended to suspend marketing authorisations for hydroxyethyl starch. This comment critically evaluates the line of arguments. Basically, the only indication for a colloid is intravascular hypovolemia. Crystalloid use appears reasonable to compensate ongoing extracellular losses beyond. In the hemodynamically instable patient this leads to the distinction between an initial resuscitation phase where colloids might be indicated and a crystalloidal maintenance phase thereafter. It is important to bear this in mind when reevaluating the studies the EMA referred to in the context of its recent decision: i) VISEP compared ringer's lactate to 10% HES 200/0.5 in septic patients and found an increased incidence of renal failure in HES receivers. Unfortunately, study treatment was started only after initial stabilization with HES, randomizing hemodynamically stable patients into a rational (crystalloids) and an irrational (high dose starch until ICU discharge) maintenance treatment. ii) 6S compared ringer's acetate to 6% HES 130/0.42 for fluid resuscitation in septic patients and found an increased need of renal replacement therapy and a higher mortality in the HES group. However, patients of both groups were again randomized only after initial stabilization with colloids, the actual comparison was, therefore, again rational vs. irrational. Beyond that, the documentation is partly fragmentary, leaving many important questions around the fate of the patients unanswered. iii) CHEST randomized ICU patients to receive saline or 6% HES 130/0.4 for fluid resuscitation. Actually, despite partly discussed in a different way, this trial showed no relevant differences in outcome. In all, two studies showed what happens to septic patients if starches are used in a way we do not observe in daily practice. The third one actually proves their safety. The benefit of perioperative goal

  12. Novel polymer blends with thermoplastic starch

    NASA Astrophysics Data System (ADS)

    Taghizadeh, Ata

    A new class of polymers known as "bioplastics" has emerged and is expanding rapidly. This class consists of polymers that are either bio-based or biodegradable, or both. Among these, polysaccharides, namely starch, are of great interest for several reasons. By gelatinizing starch via plasticizers, it can be processed in the same way as thermoplastic polymers with conventional processing equipment. Hence, these bio-based and biodegradable plastics, with their low source and refinery costs, as well as relatively easy processability, have made them ideal candidates for incorporation into various current plastic products. Four different plasticizers have been chosen here for gelatinization of thermoplastic starch (TPS): glycerol, sorbitol, diglycerol and polyglycerol, with the latter two being used for the first time in such a process. Two methodological categories are used. The first involves a calorimetric method (Differential Scanning Calorimetry) as well as optical microscopy; these are "static" methods where no shear is applied A wide range of starch/water/plasticizer compositions were prepared to explore the gelatinization regime for each plasticizer. The onset and conclusion gelatinization temperatures for sorbitol and glycerol were found to be in the same vicinity, while diglycerol and polyglycerol showed significantly higher transition temperatures. The higher molecular weight and viscosity of polyglycerol allow this transition to occur at an even higher temperature than with diglycerol. This is due to the increase in molecular weight and viscosity of the two new plasticizers, as well as their significant decrease in water solubility. It is demonstrated that the water/plasticizer ratio has a pronounced effect on gelatinization temperatures. When plasticizer content was held constant and water content was increased, it was found that the gelatinization temperature decreased for all the plasticizers. Meanwhile, when the water content was held constant and the

  13. Novel polymer blends with thermoplastic starch

    NASA Astrophysics Data System (ADS)

    Taghizadeh, Ata

    A new class of polymers known as "bioplastics" has emerged and is expanding rapidly. This class consists of polymers that are either bio-based or biodegradable, or both. Among these, polysaccharides, namely starch, are of great interest for several reasons. By gelatinizing starch via plasticizers, it can be processed in the same way as thermoplastic polymers with conventional processing equipment. Hence, these bio-based and biodegradable plastics, with their low source and refinery costs, as well as relatively easy processability, have made them ideal candidates for incorporation into various current plastic products. Four different plasticizers have been chosen here for gelatinization of thermoplastic starch (TPS): glycerol, sorbitol, diglycerol and polyglycerol, with the latter two being used for the first time in such a process. Two methodological categories are used. The first involves a calorimetric method (Differential Scanning Calorimetry) as well as optical microscopy; these are "static" methods where no shear is applied A wide range of starch/water/plasticizer compositions were prepared to explore the gelatinization regime for each plasticizer. The onset and conclusion gelatinization temperatures for sorbitol and glycerol were found to be in the same vicinity, while diglycerol and polyglycerol showed significantly higher transition temperatures. The higher molecular weight and viscosity of polyglycerol allow this transition to occur at an even higher temperature than with diglycerol. This is due to the increase in molecular weight and viscosity of the two new plasticizers, as well as their significant decrease in water solubility. It is demonstrated that the water/plasticizer ratio has a pronounced effect on gelatinization temperatures. When plasticizer content was held constant and water content was increased, it was found that the gelatinization temperature decreased for all the plasticizers. Meanwhile, when the water content was held constant and the

  14. Impact of Oxidative Stress on Ascorbate Biosynthesis in Chlamydomonas via Regulation of the VTC2 Gene Encoding a GDP-l-galactose Phosphorylase*

    PubMed Central

    Urzica, Eugen I.; Adler, Lital N.; Page, M. Dudley; Linster, Carole L.; Arbing, Mark A.; Casero, David; Pellegrini, Matteo; Merchant, Sabeeha S.; Clarke, Steven G.

    2012-01-01

    The l-galactose (Smirnoff-Wheeler) pathway represents the major route to l-ascorbic acid (vitamin C) biosynthesis in higher plants. Arabidopsis thaliana VTC2 and its paralogue VTC5 function as GDP-l-galactose phosphorylases converting GDP-l-galactose to l-galactose-1-P, thus catalyzing the first committed step in the biosynthesis of l-ascorbate. Here we report that the l-galactose pathway of ascorbate biosynthesis described in higher plants is conserved in green algae. The Chlamydomonas reinhardtii genome encodes all the enzymes required for vitamin C biosynthesis via the l-galactose pathway. We have characterized recombinant C. reinhardtii VTC2 as an active GDP-l-galactose phosphorylase. C. reinhardtii cells exposed to oxidative stress show increased VTC2 mRNA and l-ascorbate levels. Genes encoding enzymatic components of the ascorbate-glutathione system (e.g. ascorbate peroxidase, manganese superoxide dismutase, and dehydroascorbate reductase) are also up-regulated in response to increased oxidative stress. These results indicate that C. reinhardtii VTC2, like its plant homologs, is a highly regulated enzyme in ascorbate biosynthesis in green algae and that, together with the ascorbate recycling system, the l-galactose pathway represents the major route for providing protective levels of ascorbate in oxidatively stressed algal cells. PMID:22393048

  15. Characteristics of taro (Colocasia esculenta) starches planted in different seasons and their relations to the molecular structure of starch.

    PubMed

    Lu, Ting-Jang; Lin, Jheng-Hua; Chen, Jia-Ci; Chang, Yung-Ho

    2008-03-26

    Physico-chemical properties and molecular structure of starches from three cultivars (Dog hoof, Mein, and KS01) of taro tubers planted in summer, winter, and spring were investigated. The effects of the planting season on the physico-chemical properties and the molecular structure of starch were determined, and the relations between the physico-chemical properties and the molecular structure of starch are discussed. Results indicate that taro starches from tubers planted in summer had the largest granule size, a low uniformity of gelatinization, and a high tendency to swell and collapse when heated in water. Taro starch planted in summer also showed an elasticity during gelatinization that was higher than that of starches planted in the other seasons. In addition to the planting season and the variety, rheological and pasting properties of taro starches studied are influenced not only by the amylose content but also by the chain-length distribution of amylopectin, whereas swelling power and solubility only depend on the amylose content of starch. Taro starch with relatively high amylose content, high short-to-long-chain ratio, and long average chain length of long-chain fraction of amylopectin displayed high elasticity and strong gel during heating.

  16. Tartary buckwheat (Fagopyrum tataricum Gaertn.) starch, a side product in functional food production, as a potential source of retrograded starch.

    PubMed

    Gao, Jinfeng; Kreft, Ivan; Chao, Guimei; Wang, Ying; Liu, Xiaojin; Wang, Li; Wang, Pengke; Gao, Xiaoli; Feng, Baili

    2016-01-01

    A starch rich fraction is a side product in Tartary buckwheat processing. This study investigated the fractions that are of technological and nutritional interest. Tartary buckwheat starch granules had a diameter of 3-14 μm, and presented a typical type "A" X-ray diffraction pattern. They contained nearly 39.0% amylose. The solubility of Tartary buckwheat starch was much lower at 70-90 °C (ranging within 9.9-10.4% at 90 °C) than that in maize (up to 49.3%) and potato (up to 85.0%) starch. The starch of one variety of Tartary buckwheat had significantly lower solubility at 70 °C and 80 °C than that of common buckwheat. The starch peak viscosity and breakdown were higher and pasting time was shorter in Tartary buckwheat than in that of the starch of common buckwheat. Tartary buckwheat starch had unique pasting and physicochemical properties, and is thereby capable of being exploited as a suitable raw material of retrograded starch in food processing.

  17. The deposition and characterization of starch in Brachypodium distachyon

    PubMed Central

    Tanackovic, Vanja; Svensson, Jan T.; Jensen, Susanne L.; Buléon, Alain; Blennow, Andreas

    2014-01-01

    Brachypodium distachyon is a non-domesticated cereal. Nonetheless, Brachypodium was recently introduced as a model plant for temperate cereals. This study compares grain starch metabolism in Brachypodium and barley (Hordeum vulgare). In Brachypodium, we identified and annotated 28 genes involved in starch metabolism and identified important motifs including transit peptides and putative carbohydrate-binding modules (CBMs) of the families CBM20, CBM45, CBM48, and CBM53. Starch content was markedly lower in Brachypodium grains (12%) compared to barley grains (47%). Brachypodium starch granules were doughnut shaped and bimodally distributed into distinct small B-type (2.5–10 µm) and very small C-type (0.5–2.5 µm) granules. Large A-type granules, typical of cereals, were absent. Starch-bound phosphate, important for starch degradation, was 2-fold lower in Brachypodium compared with barley indicating different requirements for starch mobilization. The amylopectin branch profiles were similar and the amylose content was only slightly higher compared with barley cv. Golden Promise. The crystallinity of Brachypodium starch granules was low (10%) compared to barley (20%) as determined by wide-angle X-ray scattering (WAXS) and molecular disorder was confirmed by differential scanning calorimetry (DSC). The expression profiles in grain for most genes were distinctly different for Brachypodium compared to barley, typically showing earlier decline during the course of development, which can explain the low starch content and differences in starch molecular structure and granule characteristics. High transitory starch levels were observed in leaves of Brachypodium (2.8% after 14h of light) compared to barley (1.9% after 14h of light). The data suggest important pre-domesticated features of cereals. PMID:25056772

  18. Relationships among Genetic, Structural, and Functional Properties of Rice Starch.

    PubMed

    Kong, Xiangli; Chen, Yaling; Zhu, Ping; Sui, Zhongquan; Corke, Harold; Bao, Jinsong

    2015-07-15

    We determined the relationships among the structural properties, in vitro digestibility, and genetic factors in starches of 14 rice cultivars. Weight-based chain-length distributions in amylopectin ranged from 18.07% to 24.71% (fa, DP 6-12), 45.01% to 55.67% (fb1, DP 13-24), 12.72% to 14.05% (fb2, DP 25-36), and 10.80 to 20.72% (fb3, DP > 36), respectively. The contents of rapidly digestible starch (RDS), slowly digestible starch (SDS), and resistant starch (RS) ranged from 78.5% to 87.5%, 1.2% to 6.0%, and 10.1% to 18.0%, respectively. AAC was negatively correlated with RDS content but positively correlated with RS content in rice starch. The proportion of short chains in amylopectin, i.e. the amount of fraction IIa (FrIIa) fractionated by gel permeation chromatography (GPC), was positively correlated with RDS. Starch synthase IIa (SSIIa) gene controlled the degree of crystallinity, the amount of fa chains of amylopectin. SSIIIa gene controlled the amount of fb1 chains. Wx gene controlled the FrI, FrIIa, RDS, and RS. Starch debranching enzyme isoamylase II (ISA2) gene also controlled the RDS, which may suggest that RDS was also affected by amylopectin structure, although no correlation between them was found. This study indicated that genetics (i.e., starch biosynthesis related genes) controlled the structural properties of starch, and both amylose content and amylopectin fine structure determined functional properties of rice starch (i.e., the digestion), each in a different way. Understanding the "genetics-structure-function" relationships in rice starches will assist plant breeders and food processors in developing new rice varieties and functional foods.

  19. Characterization of starch films containing starch nanoparticles. Part 2: viscoelasticity and creep properties.

    PubMed

    Shi, Ai-Min; Wang, Li-Jun; Li, Dong; Adhikari, Benu

    2013-07-25

    Starch films were successfully produced by incorporating spray dried and vacuum-freeze dried starch nanoparticles. The frequency sweep, creep-recovery behavior and time-temperature superposition (TTS) on these films were studied. All these films exhibited dominant elastic behavior (than viscous behavior) over the entire frequency range (0.1-100 rad/s). The incorporation of both types of starch nanoparticles increased the storage and loss modulus, tanδ, creep strain, creep compliance and creep rate at long time frame and reduced the recovery rate of films while the effect of different kinds of starch nanoparticles on these parameters was similar both in magnitude and trend. TTS method was successfully used to predict long time (over 20 days) creep behavior through the master curves. The addition of these nanoparticles could increase the activation energy parameter used in TTS master curves. Power law and Burger's models were capable of fitting storage and loss modulus (R(2)>0.79) and creep data (R(2)>0.96), respectively.

  20. The surface-associated proteins of wheat starch granules: suitability of wheat starch for celiac patients

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Wheat starch is used to make baked products for celiac patients in several European countries, but is avoided in the US because of uncertainty about the amounts of associated grain storage (gluten) proteins. People with celiac disease (CD) must avoid wheat, rye and barley proteins and products that...

  1. Porous starch extracted from Chinese rice wine vinasse: characterization and adsorption properties.

    PubMed

    Li, Hongyan; Jiao, Aiquan; Wei, Benxi; Wang, Yong; Wu, Chunsen; Jin, Zhengyu; Tian, Yaoqi

    2013-10-01

    Chinese rice wine vinasse (the fermentation residue after removal of the crude wine or beer) contains 20-30% residual native starch. These starches are partly hydrolyzed by amylase and glucoamylase during rice wine fermentation, indicating that it is a potential source of porous starch, which is a value-added material. In the present study, morphological, short-range order, crystalline, and thermal studies were determined to characterize the structural and chemical properties of vinasse starch. The results showed that vinasse starch granule had a rough and porous shape and was much more ordered than native starch. Vinasse starch also could tolerate a higher temperature than native starch. The water and oil adsorptive capacities of vinasse starch were 1.89 and 4.14 times higher than that of native rice starch. These results suggest that vinasse is an effective and economical source of porous starch for using as adsorbent. PMID:23850681

  2. Association of alpha-amylase and the R1 protein with starch granules precedes the initiation of net starch degradation in turions of Spirodela polyrhiza.

    PubMed

    Reimann, Rezarta; Ritte, Gerhard; Steup, Martin; Appenroth, Klaus-J

    2002-01-01

    In turions of Spirodela polyrhiza (L.) Schleiden, net degradation of storage starch is controlled by a special low fluence response of phytochrome requiring illumination for several days. This light effect has been used to study protein-starch interactions that occur prior to and during net degradation of starch. Following various pretreatments on S. polyrhiza turions, native starch granules were isolated and two fractions of starch-related proteins were distinguished: proteins enclosed within the starch particles (starch-internalized proteins) and those attached to the surface (starch-associated proteins). The pattern of starch-associated proteins as resolved by SDS-PAGE was more complex than that of starch-internalized proteins and varied depending upon the pretreatment of the turions. Two starch associated proteins were identified immunochemically as alpha-amylase (EC 3.2.1.1) and the R1 protein (Lorberth et al. (1998) Nature Biotechnology 16: 473-477). Dark-pretreatment of non-dormant turions does not induce starch net degradation. Under these conditions, alpha-amylase and R1 were bound to the surface of the starch granules. Continuous illumination with red light induces a rapid degradation of starch. Within the first 24 h of illumination the level of starch-associated alpha-amylase transiently increased and subsequently decreased rapidly. Similarly, the amount of the starch-associated R1 also decreased during illumination. The dissociation of both alpha-amylase and R1 from the starch granules preceded the decrease in starch content. However, binding of the two proteins to starch granules remained unchanged when the turions did not perform net starch degradation (as observed during continuous darkness, orthophosphate deficiency, or dormancy of the turions). Thus, during net starch degradation, so far unidentified changes are postulated to occur at the surface of the starch particles that are relevant for protein binding. This conclusion was supported by in

  3. Association of alpha-amylase and the R1 protein with starch granules precedes the initiation of net starch degradation in turions of Spirodela polyrhiza.

    PubMed

    Reimann, Rezarta; Ritte, Gerhard; Steup, Martin; Appenroth, Klaus-J

    2002-01-01

    In turions of Spirodela polyrhiza (L.) Schleiden, net degradation of storage starch is controlled by a special low fluence response of phytochrome requiring illumination for several days. This light effect has been used to study protein-starch interactions that occur prior to and during net degradation of starch. Following various pretreatments on S. polyrhiza turions, native starch granules were isolated and two fractions of starch-related proteins were distinguished: proteins enclosed within the starch particles (starch-internalized proteins) and those attached to the surface (starch-associated proteins). The pattern of starch-associated proteins as resolved by SDS-PAGE was more complex than that of starch-internalized proteins and varied depending upon the pretreatment of the turions. Two starch associated proteins were identified immunochemically as alpha-amylase (EC 3.2.1.1) and the R1 protein (Lorberth et al. (1998) Nature Biotechnology 16: 473-477). Dark-pretreatment of non-dormant turions does not induce starch net degradation. Under these conditions, alpha-amylase and R1 were bound to the surface of the starch granules. Continuous illumination with red light induces a rapid degradation of starch. Within the first 24 h of illumination the level of starch-associated alpha-amylase transiently increased and subsequently decreased rapidly. Similarly, the amount of the starch-associated R1 also decreased during illumination. The dissociation of both alpha-amylase and R1 from the starch granules preceded the decrease in starch content. However, binding of the two proteins to starch granules remained unchanged when the turions did not perform net starch degradation (as observed during continuous darkness, orthophosphate deficiency, or dormancy of the turions). Thus, during net starch degradation, so far unidentified changes are postulated to occur at the surface of the starch particles that are relevant for protein binding. This conclusion was supported by in

  4. Elucidating the evolutionary history and expression patterns of nucleoside phosphorylase paralogs (vegetative storage proteins) in Populus and the plant kingdom

    PubMed Central

    2013-01-01

    Background Nucleoside phosphorylases (NPs) have been extensively investigated in human and bacterial systems for their role in metabolic nucleotide salvaging and links to oncogenesis. In plants, NP-like proteins have not been comprehensively studied, likely because there is no evidence of a metabolic function in nucleoside salvage. However, in the forest trees genus Populus a family of NP-like proteins function as an important ecophysiological adaptation for inter- and intra-seasonal nitrogen storage and cycling. Results We conducted phylogenetic analyses to determine the distribution and evolution of NP-like proteins in plants. These analyses revealed two major clusters of NP-like proteins in plants. Group I proteins were encoded by genes across a wide range of plant taxa while proteins encoded by Group II genes were dominated by species belonging to the order Malpighiales and included the Populus Bark Storage Protein (BSP) and WIN4-like proteins. Additionally, we evaluated the NP-like genes in Populus by examining the transcript abundance of the 13 NP-like genes found in the Populus genome in various tissues of plants exposed to long-day (LD) and short-day (SD) photoperiods. We found that all 13 of the Populus NP-like genes belonging to either Group I or II are expressed in various tissues in both LD and SD conditions. Tests of natural selection and expression evolution analysis of the Populus genes suggests that divergence in gene expression may have occurred recently during the evolution of Populus, which supports the adaptive maintenance models. Lastly, in silico analysis of cis-regulatory elements in the promoters of the 13 NP-like genes in Populus revealed common regulatory elements known to be involved in light regulation, stress/pathogenesis and phytohormone responses. Conclusion In Populus, the evolution of the NP-like protein and gene family has been shaped by duplication events and natural selection. Expression data suggest that previously

  5. Association of tumour necrosis factor alpha and its receptors with thymidine phosphorylase expression in invasive breast carcinoma.

    PubMed Central

    Leek, R. D.; Landers, R.; Fox, S. B.; Ng, F.; Harris, A. L.; Lewis, C. E.

    1998-01-01

    Angiogenesis is an essential requirement for tumour growth and metastasis and is regulated by a complex network of factors produced by both stromal cells and neoplastic cells within solid tumours. The cytokine tumour necrosis factor alpha (TNF-alpha) and the enzyme thymidine phosphorylase (TP) are two factors known to promote tumour angiogenesis. We have demonstrated recently that high numbers of tumour-associated macrophages (TAMs) are significantly associated with increased tumour angiogenesis and poor prognosis in invasive carcinoma of the breast. We have also shown that TAMs are a major source of TNF-alpha in invasive breast carcinomas, and that macrophage-like stromal cells as well as tumour cells synthesize TP in such tumours. However, little is known of the factors that regulate the production or activity of these factors in the tumour microenvironment. As TNF-alpha has been shown to up-regulate TP expression in tumour cells in vitro we performed an immunohistochemical study to investigate the possibility that TNF-alpha may be involved in the regulation of TP expression by malignant breast epithelial cells in vivo. To do this, we used a cocktail of non-neutralizing monoclonal anti-TNF-alpha antibodies to visualize both TNF-alpha-expressing macrophages and TNF-alpha bound to its receptors on tumour cells and endothelial cells in a series of 93 invasive carcinomas of the breast. A semiquantitative grading system was then used to compare these staining patterns with that for TP in the same biopsies. TNF-alpha immunoreactivity was also compared with various important tumour variables known to relate to outcome in this disease (microvessel density, node status, grade, stage, receptor status and macrophage infiltration), as well as relapse-free and overall survival data for these patients. Our data show significant positive correlations between TNF-alpha bound to its receptors on tumour cells and: (1) TP protein production by tumour cells, and (2) axillary lymph

  6. Effect of hydrocolloids on functional properties of navy bean starch

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Pulses are recognized as a low-fat source of gluten-free protein, soluble fiber, B-vitamins and minerals, and their use in foods has increased in recent years. The functional properties of pulse starches have received relatively little attention, but they are important since starch is the major comp...

  7. Colloid Characteristics and Emulsifying Properties of OSA Starches

    NASA Astrophysics Data System (ADS)

    Dokić, Petar; Dokić, Ljubica; Dapčević, Tamara; Krstonošić, Veljko

    The objective of this paper was to characterize commercially available octenyl-succinate starches (OSA starches), Purity Gum 2000 and Hi-cap 100, by determining their viscous behaviour and characteristics and molecular mass, as well as to examine their emulsifying properties in corn oil-in-water emulsion system.

  8. Individual mammalian mucosal glucosidase subunits digest various starch structures differently

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Starch digestion in the human body requires two luminal enzymes,salivary and pancreatic alpha-amylase (AMY), and four small intestinal mucosal enzyme activities related to the maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI) complexes. Starch consists of two polysaccharides, amylose (AM) and ...

  9. Physicochemical properties of cassava starch oxidized by sodium hypochlorite.

    PubMed

    Garrido, Lúcia Helena; Schnitzler, Egon; Zortéa, Manoela Estefânea Boff; de Souza Rocha, Thaís; Demiate, Ivo Mottin

    2014-10-01

    In this work, cassava starch was modified by treatment with sodium hypochlorite (NaClO) at different concentrations (0.8, 2.0 and 5.0 % of active chlorine) and selected physicochemical properties of the oxidized starches were investigated. The native and modified samples were evaluated considering moisture, carboxyl content, apparent viscosity, susceptibility to syneresis, mid-infrared spectroscopy and crystallinity index. The treatment with NaClO resulted in alterations in carboxyl content of the oxidized starches that increased with increasing concentration of the oxidant. Oxidized starches also showed higher susceptibility to syneresis, as assessed by the release of liquid during freezing and thawing. Apparent viscosity analysis showed decrease in peak viscosity of the oxidized starches. X-ray diffractograms showed that the oxidation influenced the extent of cassava starch relative crystallinity found to lie between 34.4 % (native) and 39.9 % (2.0 % active chlorine). The infrared spectra are sensitive to structural changes on starch macromolecules and presented characteristic peaks as C-O-C of the six carbon glucose ring absorbs at 1,150-1,085 cm(-1) and due to axial deformation these bands changed with the crystal structure of the starch samples.

  10. Battle of the starches: Insoluble versus soluble at the refinery

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A study using the USDA starch research method has been conducted to evaluate the effects of total, insoluble, and soluble starch on raw sugar filterability and viscosity in international carbonatation refineries. Raw sugar qualities, i.e., pol, color, % invert, ash, and dextran, were also studied in...

  11. Properties of Cassava Starch Modified by Amylomaltase from Corynebacterium glutamicum.

    PubMed

    Suriyakul Na Ayudhaya, Pitcha; Pongsawasdi, Piamsook; Laohasongkram, Kalaya; Chaiwanichsiri, Saiwarun

    2016-06-01

    Amylomaltase (α-1,4-glucanotransferase, AM; EC 2.4.1.25) from Corynebacterium glutamicum expressed in Escherichia coli was used to prepare the enzyme-modified cassava starch for food application. About 5% to 15% (w/v) of cassava starch slurries were incubated with 1, 3, or 5 units of amylomaltase/g starch. Apparent amylose, amylopectin chain length distribution, thermal properties, freeze-thaw stability, thermo-reversibility, and gel strength of the obtained modified starches were measured. The apparent amylose content and retrogradation enthalpy were lower, whereas the retrogradation temperatures, freeze-thaw stability, and thermo-reversibility were higher than those of the native cassava starch. However, when amylomaltase content was increased to 20 units of amylomaltase/g starch and for 24 h, the modified starch showed an improvement in the thermo-reversibility property. When used in panna cotta, the gel strength of the sample using the 20 units/24 h modified cassava starch was similar to that of using gelatin. PMID:27105125

  12. Physicochemical properties of cassava starch oxidized by sodium hypochlorite.

    PubMed

    Garrido, Lúcia Helena; Schnitzler, Egon; Zortéa, Manoela Estefânea Boff; de Souza Rocha, Thaís; Demiate, Ivo Mottin

    2014-10-01

    In this work, cassava starch was modified by treatment with sodium hypochlorite (NaClO) at different concentrations (0.8, 2.0 and 5.0 % of active chlorine) and selected physicochemical properties of the oxidized starches were investigated. The native and modified samples were evaluated considering moisture, carboxyl content, apparent viscosity, susceptibility to syneresis, mid-infrared spectroscopy and crystallinity index. The treatment with NaClO resulted in alterations in carboxyl content of the oxidized starches that increased with increasing concentration of the oxidant. Oxidized starches also showed higher susceptibility to syneresis, as assessed by the release of liquid during freezing and thawing. Apparent viscosity analysis showed decrease in peak viscosity of the oxidized starches. X-ray diffractograms showed that the oxidation influenced the extent of cassava starch relative crystallinity found to lie between 34.4 % (native) and 39.9 % (2.0 % active chlorine). The infrared spectra are sensitive to structural changes on starch macromolecules and presented characteristic peaks as C-O-C of the six carbon glucose ring absorbs at 1,150-1,085 cm(-1) and due to axial deformation these bands changed with the crystal structure of the starch samples. PMID:25328206

  13. Milk glucosidase activity enables suckled pup starch digestion

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Starch requires six enzymes for digestion to free glucose: two amylases (salivary and pancreatic) and four mucosal maltase activities; sucrase-isomaltase and maltase-glucoamylase. All are deficient in suckling rodents. The objective of this study is to test (13)C-starch digestion before weaning by m...

  14. Biosysthesis of Corn Starch Palmitate by Lipase Novozym 435

    PubMed Central

    Xin, Jia-Ying; Wang, Yan; Liu, Tie; Lin, Kai; Chang, Le; Xia, Chun-Gu

    2012-01-01

    Esterification of starch was carried out to expand the usefulness of starch for a myriad of industrial applications. Lipase B from Candida antarctica, immobilized on macroporous acrylic resin (Novozym 435), was used for starch esterification in two reaction systems: micro-solvent system and solvent-free system. The esterification of corn starch with palmitic acid in the solvent-free system and micro-solvent system gave a degree of substitution (DS) of 1.04 and 0.0072 respectively. Esterification of corn starch with palmitic acid was confirmed by UV spectroscopy and IR spectroscopy. The results of emulsifying property analysis showed that the starch palmitate with higher DS contributes to the higher emulsifying property (67.6%) and emulsion stability (79.6%) than the native starch (5.3% and 3.9%). Modified starch obtained by esterification that possesses emulsifying properties and has long chain fatty acids, like palmitic acid, has been widely used in the food, pharmaceutical and biomedical applications industries. PMID:22837690

  15. Films based on oxidized starch and cellulose from barley.

    PubMed

    El Halal, Shanise Lisie Mello; Colussi, Rosana; Deon, Vinícius Gonçalves; Pinto, Vânia Zanella; Villanova, Franciene Almeida; Carreño, Neftali Lenin Villarreal; Dias, Alvaro Renato Guerra; Zavareze, Elessandra da Rosa

    2015-11-20

    Starch and cellulose fibers were isolated from grains and the husk from barley, respectively. Biodegradable films of native starch or oxidized starches and glycerol with different concentrations of cellulose fibers (0%, 10% and 20%) were prepared. The films were characterized by morphological, mechanical, barrier, and thermal properties. Cellulose fibers isolated from the barley husk were obtained with 75% purity and high crystallinity. The morphology of the films of the oxidized starches, regardless of the fiber addition, was more homogeneous as compared to the film of the native starch. The addition of cellulose fibers in the films increased the tensile strength and decreased elongation. The water vapor permeability of the film of oxidized starch with 20% of cellulose fibers was lower than the without fibers. However the films with cellulose fibers had the highest decomposition with the initial temperature and thermal stability. The oxidized starch and cellulose fibers from barley have a good potential for use in packaging. The addition of cellulose fibers in starch films can contribute to the development of films more resistant that can be applied in food systems to maintain its integrity.

  16. Enzyme-Catalyzed Regioselective Modification of Starch Nanoparticles

    SciTech Connect

    Chakraborty, Soma; Sahoo, Bishwabhusan; Teraoka, Iwao; Miller, Lisa M.; Gross, Richard A.

    2004-12-13

    The selective esterification of starch nanoparticles was performed using as catalyst Candida antartica Lipase B (CAL-B) in its immobilized (Novozym 435) and free (SP-525) forms. The starch nanoparticles were made accessible for acylation reactions by formation of Aerosol-OT (AOT, bis(2-ethylhexyl)sodium sulfosuccinate) stabilized microemulsions. Starch nanoparticles in microemulsions were reacted with vinyl stearate, ε-caprolactone, and maleic anhydride at 40 °C for 48 h to give starch esters with degrees of substitution (DS) of 0.8, 0.6, and 0.4, respectively. Substitution occurred regioselectively at the C-6 position of the glucose repeat units. Infrared microspectroscopy (IRMS) revealed that AOT-coated starch nanoparticles diffuse into the outer 50 μm shell of catalyst beads. Thus, even though CAL-B is immobilized within a macroporous resin, CAL-B is sufficiently accessible to the starch nanoparticles. When free CAL-B was incorporated along with starch within AOT-coated reversed micelles, CAL-B was also active and catalyzed the acylation with vinyl stearate (24 h, 40 °C) to give DS = 0.5. After removal of surfactant from the modified starch nanoparticles, they were dispersed in DMSO or water and were shown to retain their nanodimensions.

  17. Potential Targets for Seed Improvement through Bioengineering Starch Synthesis Pathways

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Starch is the principle carbohydrate in the food chain and is a renewable and biodegradable polymer widely used in the food, beverage, paper, textile, and livestock feed industries. It is the major component of the harvestable organs in many agronomic plants. The quantity and quality of starch thus ...

  18. Optimization of cationic amino starch synthesis using biogenic amines.

    PubMed

    Anthony, Renil John; Sims, Ronald C

    2013-11-01

    Harvesting microalgae presents a challenge in selecting the most economical method for low cost algal bioproducts. Previous studies have shown coagulation-flocculation to be the most efficient method for large scale microalgae harvesting. This study focused on modifying native potato starch with biogenic amines and optimizing the reaction parameters. Such modification rendered the starch cationic, with an ability to destabilize microalgae suspensions or colloids. The effect of time, temperature, and reactant concentrations on the zeta potential of the cationic amino starch was studied. Biogenic amines including putrescine, histamine, cadaverine, and tyramine were selected for study based on the number of nitrogen groups in their structure. Zeta potential for histamine cationic amino starch was significantly higher (+9.0±2.0 mV) at lower reaction temperatures, regardless of the amine to starch ratio and reaction time intervals. Putrescine, cadaverine, and tyramine cationic amino starches exhibited significantly higher zeta potential values (13.76±3.60, 6.81±1.64, and 5.68±1.60 mV, respectively) with amine to starch ratio higher than reaction stoichiometry, irrespective of reaction temperature or time intervals. This optimization study has presented a basis for designing reaction conditions for the synthesis of cationic amino starch from an inhomogeneous mix of biogenic amines derived from waste sources.

  19. Flocculation of Kaolin by Waxy Maize Starch Phosphates

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Waxy maize starch phosphates were tested as flocculants in order to determine if they have the potential to replace petroleum-based polymer flocculants currently used commercially. Phosphorylation was carried out by drying heating of starches and sodium orthophosphates at 140 deg C for 4 h. Native...

  20. Flocculation of Kaolin by Waxy Maize Starch Phosphates

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Waxy maize starch phosphates were tested as flocculants in order to determine if they have the potential to replace petroleum-based polymer flocculants currently used commercially. Phosphorylation was carried out by dry heating of starches and sodium orthophosphates at 140 degrees C for 4 hours. N...