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1.
Arch Biochem Biophys ; 562: 9-21, 2014 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-25107532

RESUMO

To investigate the functional properties of 10 α-glucan branching enzymes (BEs) from various sources, we determined the chain-length distribution of BE enzymatic products and their phosphorylase-limit dextrins (Φ-LD). All BEs could be classified into either of the three rice BE isozymes: OsBEI, OsBEIIa, or OsBEIIb. Escherichia coli BE (EcoBE) had the same enzymatic properties as OsBEI, while Synechococcus elongatus BE (ScoBE) and Chlorella kessleri BE (ChlBE) had BEIIb-type properties. Human BE (HosBE), yeast BE (SacBE), and two Porphyridium purpureum BEs (PopBE1 and PopBE2) exhibited the OsBEIIa-type properties. Analysis of chain-length profile of Φ-LD of the BE reaction products revealed that EcoBE, ScoBE, PopBE1, and PopBE2 preferred A-chains as acceptors, while OsBEIIb used B-chains more frequently than A-chains. Both EcoBE and ScoBE specifically formed the branch linkages at the third glucose residue from the reducing end of the acceptor chain. The present results provide evidence for the first time that great variation exists as to the preference of BEs for their acceptor chain, either A-chain or B-chain. In addition, EcoBE and ScoBE recognize the location of branching points in their acceptor chain during their branching reaction. Nevertheless, no correlation exists between the primary structure of BE proteins and their enzymatic characteristics.


Assuntos
Enzima Ramificadora de 1,4-alfa-Glucana/química , Glucanos/química , Amilopectina/química , Chlorella/enzimologia , Dextrinas/química , Escherichia coli/enzimologia , Fungos/enzimologia , Glicogênio/química , Humanos , Isoenzimas/química , Oryza/enzimologia , Fosforilases/química , Filogenia , Porphyridium/enzimologia , Proteínas Recombinantes/química , Especificidade da Espécie , Amido/química , Synechococcus/enzimologia
2.
Plant J ; 62(5): 785-95, 2010 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-20202172

RESUMO

The Arabidopsis genome has two fumarase genes, one of which encodes a protein with mitochondrial targeting information (FUM1) while the other (FUM2) does not. We show that a FUM1-green fluorescent protein fusion is directed to mitochondria while FUM2-red fluorescent protein remains in the cytosol. While mitochondrial FUM1 is an essential gene, cytosolic FUM2 is not required for plant growth. However FUM2 is required for the massive accumulation of carbon into fumarate that occurs in Arabidopsis leaves during the day. In fum2 knock-out mutants, fumarate levels remain low while malate increases, and these changes can be reversed with a FUM2 transgene. The fum2 mutant has lower levels of many amino acids in leaves during the day compared with the wild type, but higher levels at night, consistent with a link between fumarate and amino acid metabolism. To further test this relationship we grew plants in the absence or presence of nitrogen fertilizer. The amount of fumarate in leaves increased several fold in response to nitrogen in wild-type plants, but not in fum2. Malate increased to a small extent in the wild type but to a greater extent in fum2. Growth of fum2 plants was similar to that of the wild type in low nitrogen but much slower in the presence of high nitrogen. Activities of key enzymes of nitrogen assimilation were similar in both genotypes. We conclude that FUM2 is required for the accumulation of fumarate in leaves, which is in turn required for rapid nitrogen assimilation and growth on high nitrogen.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Fumarato Hidratase/metabolismo , Fumaratos/metabolismo , Nitrogênio/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , DNA Bacteriano/genética , Fumarato Hidratase/genética , Regulação da Expressão Gênica de Plantas , Técnicas de Inativação de Genes , Metaboloma , Mutagênese Insercional , Mutação , Folhas de Planta/enzimologia , Folhas de Planta/genética , RNA de Plantas/genética
3.
Plant Cell Physiol ; 50(6): 1062-74, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19380351

RESUMO

In monocots, starch branching enzyme II (BEII) was functionally differentiated into BEIIa and BEIIb after separation from the dicots, and in cereals BEIIb plays a distinct role in amylopectin biosynthesis in the endosperm. The present study was conducted to examine to what extent a green algal BEII has an overlapping function with BEIIb in starch biosynthesis by introducing the Chlorella BEII gene into an amylose-extender (ae) mutant of rice. Chlorella BEII was found to complement the contribution of the rice endosperm BEIIb to the structures of amylopectin and starch granules because these mutated phenotypes were recovered almost completely to those of the wild type by the expression of Chlorella BEII. When the recombinant BE enzymes were incubated with the rice ae amylopectin, the branching pattern of Chlorella BEII was much more similar to that of rice BEIIb rather than rice BEIIa. Detailed analyses of BE reaction products suggests that BEIIb and Chlorella BEII only transfer chains with a degree of polymerization (DP) of 6 and 7, whereas BEIIa preferably transfers short chains with a DP of about 6-11. These results show that the Chlorella BEII is functionally similar to rice BEIIb rather than BEIIa.


Assuntos
Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Chlorella/enzimologia , Oryza/enzimologia , Proteínas de Plantas/metabolismo , Enzima Ramificadora de 1,4-alfa-Glucana/genética , Amilopectina/biossíntese , Chlorella/genética , Regulação da Expressão Gênica de Plantas , Oryza/genética , Fenótipo , Filogenia , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/genética
4.
Mar Biotechnol (NY) ; 9(2): 192-202, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17160635

RESUMO

Red algae are widely known to produce floridean starch but it remains unclear whether the molecular structure of this algal polyglucan is distinct from that of the starch synthesized by vascular plants and green algae. The present study shows that the unicellular species Porphyridium purpureum R-1 (order Porphyridiales, class Bangiophyceae) produces both amylopectin-type and amylose-type alpha-polyglucans. In contrast, Cyanidium caldarium (order Porphyridiales, class Bangiophyceae) synthesizes glycogen-type polyglucan, but not amylose. Detailed analysis of alpha-1,4-chain length distribution of P. purpureum polyglucan suggests that the branched polyglucan has a less ordered structure, referred to as semi-amylopectin, as compared with amylopectin of rice endosperm having a tandem-cluster structure. The P. purpureum linear amylose-type polyglucan, which has a lambda(max) of 630 nm typical of amylose-iodine complex and is resistant to Pseudomonas isoamylase digestion, accounts for less than 10% of the total polyglucans. We produced and isolated a cDNA encoding a granule-bound starch synthase (GBSS)-type protein of P. purpureum, which is probably the approximately 60-kDa protein bound tightly to the starch granules, resembling the amylose-synthesizing GBSS protein of green plants. The present investigation indicates that the class Bangiophyceae includes species producing both semi-amylopectin and amylose, and species producing glycogen alone.


Assuntos
Amilopectina/química , Amilose/química , Glicogênio/química , Rodófitas/fisiologia , Proteínas de Algas/análise , Sequência de Aminoácidos , Glucanos/química , Glucanos/isolamento & purificação , Dados de Sequência Molecular , Filogenia , Porphyridium/química , Porphyridium/fisiologia , Rodófitas/química , Alinhamento de Sequência , Sintase do Amido/análise , Sintase do Amido/química , Sintase do Amido/genética , Difração de Raios X
5.
J Exp Bot ; 56(422): 3229-44, 2005 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16275672

RESUMO

A comprehensive analysis of the transcript levels of genes which encode starch-synthesis enzymes is fundamental for the assessment of the function of each enzyme and the regulatory mechanism for starch biosynthesis in source and sink organs. Using quantitative real-time RT-PCR, an examination was made of the expression profiles of 27 rice genes encoding six classes of enzymes, i.e. ADPglucose pyrophosphorylase (AGPase), starch synthase, starch branching enzyme, starch debranching enzyme, starch phosphorylase, and disproportionating enzyme in developing seeds and leaves. The modes of gene expression were tissue- and developmental stage-specific. Four patterns of expression in the seed were identified: group 1 genes, which are expressed very early in grain formation and are presumed to be involved in the construction of fundamental cell machineries, de novo synthesis of glucan primers, and initiation of starch granules; group 2 genes, which are highly expressed throughout endosperm development; group 3 genes, which have transcripts that are low at the onset but which rise steeply at the start of starch synthesis in the endosperm and are thought to play essential roles in endosperm starch synthesis; and group 4 genes, which are expressed scantly, mainly at the onset of grain development, and might be involved in synthesis of starch in the pericarp. The methodology also revealed that the defect in the cytosolic AGPase small subunit2b (AGPS2b) transcription from the AGPS2 gene in endosperm sharply enhanced the expressions of endosperm and leaf plastidial AGPS1, the endosperm cytosolic AGPase large subunit2 (AGPL2), and the leaf plastidial AGPL1.


Assuntos
Regulação da Expressão Gênica de Plantas , Oryza/genética , Amido/biossíntese , Amilopectina/metabolismo , Arabidopsis/genética , Perfilação da Expressão Gênica , Genes de Plantas , Glucose-1-Fosfato Adenililtransferase/classificação , Glucose-1-Fosfato Adenililtransferase/metabolismo , Glucosiltransferases/classificação , Glucosiltransferases/metabolismo , Família Multigênica , Mutação , Oryza/enzimologia , Oryza/crescimento & desenvolvimento , Filogenia , Folhas de Planta/enzimologia , Folhas de Planta/genética , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , Sementes/enzimologia , Sementes/genética , Sementes/crescimento & desenvolvimento , Solanum tuberosum/genética , Zea mays/genética
6.
Plant Mol Biol ; 58(2): 213-27, 2005 May.
Artigo em Inglês | MEDLINE | ID: mdl-16027975

RESUMO

Four amino acids were variable between the 'active' indica-type and 'inactive' japonica-type soluble starch synthase IIa (SSIIa) of rice plants; Glu-88 and Gly-604 in SSIIa of indica-cultivars IR36 and Kasalath were replaced by Asp-88 and Ser-604, respectively, in both japonica cultivars Nipponbare and Kinmaze SSIIa, whereas Val-737 and Leu-781 in indica SSIIa were replaced by Met-737 in cv. Nipponbare and Phe-781 in cv. Kinmaze SSIIa, respectively. The SSIIa gene fragments shuffling experiments revealed that Val-737 and Leu-781 are essential not only for the optimal SSIIa activity, but also for the capacity to synthesize indica-type amylopectin. Surprisingly, however, a combination of Phe-781 and Gly-604 could restore about 44% of the SSIIa activity provided that Val-737 was conserved. The introduction of the 'active' indica-type SSIIa gene enabled the japonica-type cv. Kinmaze to synthesize indica-type amylopectin. The starch in the transformed japonica rice plants exhibited gelatinization-resistant properties that are characteristic of indica-rice starch. Transformed lines expressing different levels of the IR36 SSIIa protein produced a variety of starches with amylopectin chain-length distribution patterns that correlated well with their onset temperatures of gelatinization. The present study confirmed that the SSIIa activity determines the type of amylopectin structure of rice starch to be either the typical indica-type or japonica-type, by playing a specific role in the synthesis of the long B(1) chains by elongating short A and B(1) chains, notwithstanding the presence of functional two additional SSII genes, a single SSI gene, two SSIII genes, and two SSIV genes in rice plants.


Assuntos
Aminoácidos/genética , Amilopectina/metabolismo , Oryza/genética , Proteínas de Plantas/genética , Sintase do Amido/genética , Amido/metabolismo , Substituição de Aminoácidos , Clonagem Molecular , DNA Complementar/química , DNA Complementar/genética , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Dados de Sequência Molecular , Oryza/enzimologia , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sementes/enzimologia , Sementes/genética , Sementes/metabolismo , Análise de Sequência de DNA , Especificidade da Espécie , Sintase do Amido/metabolismo
7.
Microbiology (Reading) ; 146 ( Pt 11): 2937-2946, 2000 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-11065372

RESUMO

Streptomyces coelicolor A3(2) strain M145 has eight chitinase genes scattered on the chromosome: six genes for family 18 (chiA, B, C, D, E and H) and two for family 19 (chiF and G). In this study, the expression and regulation of these genes were investigated. The transcription of five of the genes (chiA, B, C, D and F) was induced in the presence of colloidal chitin while that of the other three genes (chiE, G and H) was not. The transcripts of the five induced chi genes increased and reached their maximum at 4 h after the addition of colloidal chitin, all showing the same temporal patterns. The induced levels of the transcripts of chiB were significantly lower than those of the other four genes. Dynamic analysis of the transcripts of the chi genes indicated that chiA and chiC were induced more strongly than chiD and chiF. Addition of chitobiose also induced transcription of the chi genes, but significantly earlier than did colloidal chitin. When cells were cultured in the presence of colloidal chitin, an exponential increase of chitobiose concentration in the culture supernatant was observed prior to the induced transcription of the chi genes. This result, together with the immediate effect of chitobiose on the induction, suggests that chitobiose produced from colloidal chitin is involved in the induction of transcription of the chi genes. The transcription of the five chi genes was repressed by glucose. This repression was apparently mediated by the glucose kinase gene glkA.


Assuntos
Quitinases/genética , Genes Bacterianos , Streptomyces/enzimologia , Streptomyces/genética , Proteínas de Bactérias/genética , Sequência de Bases , Quitina/metabolismo , Quitina/farmacologia , Quitinases/metabolismo , Cromossomos Bacterianos/genética , Primers do DNA/genética , DNA Bacteriano/genética , Dissacarídeos/biossíntese , Dissacarídeos/farmacologia , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Glucoquinase/genética , Glucose/farmacologia , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Homologia de Sequência do Ácido Nucleico , Transcrição Gênica/efeitos dos fármacos
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