Purification and properties of asparaginase from the testa of immature seeds of pea (Pisum sativum L.)

A K+-dependent asparaginase (E.C. 3.5.1.1.) was purified 1328-fold from the testas of immature pea seeds (Pisum sativum L., var. Bolero) and characterized. Antibodies raised against purified asparaginase cross-reacted with the putative asparaginase band in Western blot analyses of semi-purified extracts. However, for crude extracts of pea testas, a cross-reaction was obtained with at least four protein bands, one of which was asparaginase protein. Affinity-purified antibodies to the four strongest bands of crude extracts were fairly specific for the bands from which they were purified, suggesting a mixture of specific antibodies. The Mr of asparaginase was 69,000 by Sephacryl S200 chromatography and also by mobility on native PAGE relative to BSA. There was no evidence for dissociation into subunits on SDS-PAGE, suggesting a monomeric protein of Mr 69,000. Other properties include an apparent Km of 2.4 mM, pI between 4.5 and 5, and competitive inhibition by aspartate and glycine.Uma asparaginase dependente do K+ (E.C. 3.5.1.1.) foi purificada 1328 vezes a partir de testas de sementes imaturas de ervilha (Pisum sativum L., var. Bolero) e caracterizada. Anticorpos obtidos a partir da asparaginase purificada, usados como sonda em ensaios tipo Western blot, reagiram com a banda da asparaginase de extratos semi-purificados. Entretanto, em ensaios realizados com extratos brutos de testas de ervilha, uma reação foi obtida com pelo menos 4 proteínas, entre elas a da asparaginase. O uso de anticorpos purificados por afinidade pelas 4 bandas mais evidentes do extrato bruto demonstraram um alto grau de especificidade para com as bandas de proteína das quais foram purificadas, indicando uma mistura de anticorpos específicos. A posição de eluição da atividade de asparaginase em Sephacryl S-200, bem como a sua mobilidade em PAGE não-denaturante relativo à BSA, sugere uma proteína de Mr 69.000. Em análise realizada por SDS-PAGE não foi obtida nenhuma evidência para a dissociação da proteína em subunidades, sugerindo que a proteína seja monomérica com Mr 69.000. Outras propriedades incluem um Km apparente de 2,4 mM, pI entre 4,5 e 5, e inibição competitiva pelo aspartato e pela glicina.239245Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Effect of salt on the growth and metabolism of Glycine max

Soybean plants cultivated with 50, 100 and 200 mM of NaCl, revealed that root growth was less affected by salinity than shoots. Salinity led to a reduction in leaf area and an increase in water content of the roots. These factors could contribute to the adaptation of the plant, improving its hydration. Although nitrate and free amino acid levels were reduced by salt treatment in roots, protein content of leaves was not altered. Salinity led to alterations in xylem amino acid composition, with increases in Ser, Ala, Gaba and Pro and a decrease in Asn. Similar changes were seen for Asn and Ser in roots together with a much stronger increase in Gaba. It is suggested that the decline in Asn reflects its conversion to Ala and Gaba (via Glu) in the roots while the increase in Pro and Gaba could be related to the adaptation of the plant to salinity.80981

N-partitioning, nitrate reductase and glutamine synthetase activities in two contrasting varieties of maize

Com o objetivo de identificar parâmetros que possam ser utilizados em programas de melhoramento genético em milho para uso eficiente de N, duas cultivares de milho contrastantes quanto ao uso deste nutriente, Sol da Manhã NF (eficiente) e Catetão (não-eficiente), foram avaliadas em dois experimentos conduzidos no campo e em casa de vegetação, respectivamente, sob nível baixo e alto de N. Os caracteres avaliados foram: teor e conteúdo de N em diferentes partes da planta; massa seca; peso dos grãos e de diferentes partes da planta; biomassa, e atividade das enzimas nitrato redutase e glutamina sintetase. O mecanismo de translocação de N e de fotoassimilados para os grãos e a relação fonte/dreno foram importantes para diferenciar a cultivar eficiente da não-eficiente. Conteúdo de N nos grãos e total das plantas, peso do sabugo e relação peso de espiga/matéria seca foram importantes para caracterizar a cultivar Sol da Manhã NF eficiente no uso do N. A atividade das enzimas glutamina sintetase (reação da transferase) e nitrato redutase não foram eficientes para discriminar as cultivares em estudo.In order to identify useful parameters for maize genetic breeding programs aiming at a more efficient use of N, two maize varieties of contrasting N efficiency, Sol da Manhã NF (efficient) and Catetão (inefficient) were compared. Experiments were carried out under field and greenhouse conditions, at low and high N levels. The parameters analysed included total and relative plant and grain N content, biomass and the activities of nitrate reductase and glutamine synthetase in different parts of the plant. It was found that the translocation efficiency of N and photoassimilates to the developing seeds and the source-sink relations were significantly different for the two varieties. N content of the whole plant and grain, cob weight and the relative ear dry weight were useful parameters for characterizing the variety Sol da Manhã NF as to its efficient use of N. Enzymes activity of glutamine synthetase (transferase reaction) and nitrate reductase did not differ among the varieties

Evaluation of nitrogen fertilizer and inoculation with diazotrophic bacteria on the biochemical behavior of the maize cultivar Nitroflint

Com o objetivo de verificar o efeito da adubação nitrogenada e da inoculação de bactérias diazotróficas no comportamento genético e bioquímico da cultivar de milho (Zea mays L.) Nitroflint, foram realizados dois experimentos; o primeiro no campo e o segundo em casa de vegetação. O delineamento experimental foi de blocos ao acaso com três repetições e dois fatores de avaliação: inoculação de bactérias diazotróficas (com e sem) e adubação nitrogenada (100 kg/ha de N e 10 kg/ha de N). No primeiro experimento, os caracteres avaliados foram: produção de grãos, nitrogênio total acumulado na planta (parte aérea) e no grão, atividade das enzimas nitrato redutase e glutamina sintetase. No segundo, avaliou-se o peso da planta (peso fresco) e a atividade das enzimas glutamina sintetase pelo método da transferase e sintetase, e nitrato redutase, determinada na folha e na raiz. Também foi realizada a contagem das bactérias diazotróficas na parte aérea e na raiz em três meios de cultivo: LGI (semi-seletivo para Azospirillum amazonense), JNFb (Herbaspirillum spp.) e NFb (Azospirillum spp.). Os resultados do primeiro experimento mostraram efeito da adubação nitrogenada no nitrogênio total e produção de grãos. No segundo experimento houve efeito da adubação em todos os caracteres avaliados e a inoculação apresentou efeito na atividade da enzima glutamina sintetase na raiz a partir da reação da transferase. Houve correlação da atividade da glutamina sintetase na raiz com o crescimento bacteriano no meio LGI.The evaluation of nitrogen fertilizer and inoculation with diazotrophic bacteria on the genetic and biochemical behavior of the maize (Zea mays L.) cultivar Nitroflint were performed in two experiments, one in the field and the other in greenhouse. The experimental set up consisted of a randomized blocks design with three replications and two evaluation factors, inoculation (or not) with diazotrophic bacteria and nitrogen fertilizer levels (100 kg/ha of N and 10 kg/ha of N). In the field experiment the following parameters were measured: grain production, total plant nitrogen (above ground), grain nitrogen and activities of the enzymes nitrate reductase and glutamine synthetase. In the second experiment (greenhouse), fresh weight of the plant, glutamine synthetase (transferase and synthetase) and nitrate reductase activities were determined for both leaf and root tissue. A bacterial count was made in three media, denominated LGI (semi-selective for Azospirillum amazonense), JNFb (Herbaspirillum spp.) and NFb (Azospirillum spp.) for the shoot and the root. Results of the field experiment showed an effect of N fertilizer on total N and grain production. The second experiment (greenhouse) showed an effect of N fertilizer on all parameters measured and inoculation was effective on glutamine synthetase activity of the root by transferase reaction. A correlation was obtained between root glutamine synthetase activity and bacterial growth in LGI

Distribuição de aminoácidos solúveis em endospermas mutantes de milho

A principal fonte de proteínas vegetais para alimentação humana e animal é fornecida pelos grãos de cereais e leguminosas. O conteúdo de aminoácidos solúveis totais foi determinado por HPLC em endospermas de milho (Zea mays L.) normal e mutantes opaco e floury. A concentração de aminoácidos solúveis totais variou entre os mutantes. Os mutantes o11 e o13 se destacaram com médias superiores, enquanto que mutantes o10, fl3 e fl1 apresentaram as menores médias. De maneira geral, para a concentração total de aminoácidos solúveis, os grãos dos mutantes foram similares aos seus tipos selvagens com exceção dos mutantes o11 e fl1 sendo que o primeiro apresentou valor maior que seu tipo selvagem W22, enquanto que o fl1 teve valor menor que o Oh43. Para metionina, os mutantes o2 e o11 e o tipo selvagem Oh43 apresentaram as mais altas concentrações deste aminoácido. Valores similares foram observados entre os mutantes e os tipos selvagens para concentração de outros aminoácidos analisados, tais como lisina e treonina. As altas concentrações sugeridas originalmente para estes mutantes devem ser devidas aos níveis destes aminoácidos incorporados nas proteínas de reserva, mas não na forma solúvel.For human nutrition the main source of vegetable proteins are cereal and legume seeds. The content of total soluble amino acids in mature endosperm of wild-type, opaque and floury maize (Zea mays L.) mutants were determined by HPLC. The total absolute concentration of soluble amino acids among the mutants varied depending on the mutant. The o11 and o13 mutants exhibited the highest average content, whereas o10, fl3 and fl1 exhibited the lowest average content. In general, the mutants exhibited similar concentrations of total soluble amino acids when compared to the wild-type lines, with the clear exception of mutants o11 and fl1, with the o11 mutant exhibiting a higher concentration of total soluble amino acids when compared to its wild-type counterpart W22 and the fl1 mutant a lower concentration when compared to its wild-type counterpart Oh43. For methionine, the mutants o2 and o11 and wild-type Oh43 exhibited the highest concentrations of this amino acid. Significant differences were not observed between mutants for other amino acids such as lysine and threonine. The high lysine concentrations obtained originally for these mutants may be due to the amino acids incorporated into storage proteins, but not those present in the soluble form

Distribuição de aminoácidos solúveis em endospermas mutantes de milho

For human nutrition the main source of vegetable proteins are cereal and legume seeds. The content of total soluble amino acids in mature endosperm of wild-type, opaque and floury maize (Zea mays L.) mutants were determined by HPLC. The total absolute concentration of soluble amino acids among the mutants varied depending on the mutant. The o11 and o13 mutants exhibited the highest average content, whereas o10, fl3 and fl1 exhibited the lowest average content. In general, the mutants exhibited similar concentrations of total soluble amino acids when compared to the wild-type lines, with the clear exception of mutants o11 and fl1, with the o11 mutant exhibiting a higher concentration of total soluble amino acids when compared to its wild-type counterpart W22 and the fl1 mutant a lower concentration when compared to its wild-type counterpart Oh43. For methionine, the mutants o2 and o11 and wild-type Oh43 exhibited the highest concentrations of this amino acid. Significant differences were not observed between mutants for other amino acids such as lysine and threonine. The high lysine concentrations obtained originally for these mutants may be due to the amino acids incorporated into storage proteins, but not those present in the soluble form.A principal fonte de proteínas vegetais para alimentação humana e animal é fornecida pelos grãos de cereais e leguminosas. O conteúdo de aminoácidos solúveis totais foi determinado por HPLC em endospermas de milho (Zea mays L.) normal e mutantes opaco e floury. A concentração de aminoácidos solúveis totais variou entre os mutantes. Os mutantes o11 e o13 se destacaram com médias superiores, enquanto que mutantes o10, fl3 e fl1 apresentaram as menores médias. De maneira geral, para a concentração total de aminoácidos solúveis, os grãos dos mutantes foram similares aos seus tipos selvagens com exceção dos mutantes o11 e fl1 sendo que o primeiro apresentou valor maior que seu tipo selvagem W22, enquanto que o fl1 teve valor menor que o Oh43. Para metionina, os mutantes o2 e o11 e o tipo selvagem Oh43 apresentaram as mais altas concentrações deste aminoácido. Valores similares foram observados entre os mutantes e os tipos selvagens para concentração de outros aminoácidos analisados, tais como lisina e treonina. As altas concentrações sugeridas originalmente para estes mutantes devem ser devidas aos níveis destes aminoácidos incorporados nas proteínas de reserva, mas não na forma solúvel.9196Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Analysis of alanine aminotransferase in various organs of soybean (Glycine max) and in dependence of different nitrogen fertilisers during hypoxic stress

Alanine aminotransferase (AlaAT) catalyses the reversible conversion of pyruvate and glutamate into alanine and oxoglutarate. In soybean, two subclasses were identified, each represented by two highly similar members. To investigate the role of AlaAT during hypoxic stress in soybean, changes in transcript level of both subclasses were analysed together with the enzyme activity and alanine content of the tissue. Moreover, the dependency of AlaAT activity and gene expression was investigated in relation to the source of nitrogen supplied to the plants. Using semi-quantitative PCR, GmAlaAT genes were determined to be highest expressed in roots and nodules. Under normal growth conditions, enzyme activity of AlaAT was detected in all organs tested, with lowest activity in the roots. Upon waterlogging-induced hypoxia, AlaAT activity increased strongly. Concomitantly, alanine accumulated. During re-oxygenation, AlaAT activity remained high, but the transcript level and the alanine content decreased. Our results show a role for AlaAT in the catabolism of alanine during the initial period of re-oxygenation following hypoxia. GmAlaAT also responded to nitrogen availability in the solution during waterlogging. Ammonium as nitrogen source induced both gene expression and enzyme activity of AlaAT more than when nitrate was supplied in the nutrient solution. The work presented here indicates that AlaAT might not only be important during hypoxia, but also during the recovery phase after waterlogging, when oxygen is available to the tissue again

Enzimologia da utilização de amidas em sementes imaturas

Tese (livre-docencia) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: As amidas glutamina e a asparagina são, reconhecidamente, importantes formas de transporte do nitrogênio em muitas plantas. Representam a fonte principal de nitrogênio para a síntese de proteína de reserva em sementes imaturas. Entretanto, nada se conhece a respeito da maneira pelo qual o nitrogênio desses aminoácidos se torna disponível para a biossíntese de outros aminoácidos protéicos. Objetivando esclarecer este ponto, foi realizada uma investigação sobre enzimas possivelmente envolvidas neste processo. O endosperma do milho foi escolhido para se estudar a utilização de glutamina, em virtude da importância deste composto no sistema de transporte dessa planta. Verificou-se que o endosperma apresentou atividade da enzima glutamina oxoglutarato amida transferase (GOGAT, E.C. 2.6.1.53), em níveis suficientemente altos para metabolizar toda glutamina translocada ao grão. A enzima apresentou um padrão de atividade bem definido durante a fase de maturação do endosperma, com um pico de atividade (Max. = 56 umoles GLU/min/endosperma) correspondente ao período de síntese mais intensa de proteína. Os dados sugerem que a glutamina pode ser metabolisada mediante amida para a posição 'alfa' do acetoglutarato. O glutamato assim formado poderia doar o nitrogênio para outros aminoácidos, mediante transaminação. Para se estudar a utilização de asparagina, foi escolhida a semente imatura de ervilha, em virtude da importância deste composto no seu sistema de transporte bem como no de outras leguminosas... Observação: O resumo, na íntegra, poderá ser visualizado no texto completo da tese digitalAbstract: It is well established that the amides glutamine and asparagine are important transport forms of nitrogen in many plants. They also represent the principal source of nitrogen for reserve protein synthesis in developing seeds. Since it is not known the nitrogen of these amino acids is made available for the biosynthesis of other protein amino acids in the developing seed, an investigation was made of enzymes possibly involved in this process. The utilization of glutamine was studied in the developing maize endosperm, in view of its importance as a transport compound in this plant. It was found that the endosperm contains glutamine: oxoglutarate amide transferase (GOGAT, E.C. 2.6.1.53) activity, at levels sufficient to metabolize all glutamine arriving via the translocation stream. The activity pattern throughout endosperm development revealed a well-defined peak (max. = 56 umoles GLU/min/endosperm) coinciding with the period of most active nitrogen accumulation. The data suggest that glutamine may be metabolized by transfer of the amide nitrogen to the 'alfa'-position of 'alfa'-ketoglutarate to form glutamate, via the GOGAT reaction, and subsequently to other amino acids via transamination reactions involving glutamate. The utilization of asparagines was studied in the developing seeds of pea, in view of its importance in the transport of nitrogen in this as well as other legumes... Note: The complete abstract is available with the full electronic digital thesis or dissertationsLivre DocênciaLivre-Docente em Biologia Funcional e Molecula

Products of anaerobic metabolism in waterlogged roots of soybean are exported in the xylem

Waterlogging leads to hypoxia of the root system. Metabolic changes occur that enable the plant to tolerate the hypoxic stress. We investigated the export of organic acids, products of anaerobic metabolism, via xylem of waterlogged soybean (Glycine max) plants. Organic acids were quantified by GC-MS and their formation via aspartate metabolism investigated using [4-C-13]aspartate. Elevated levels of malate were found together with variable amounts of other organic acids, notably lactate and succinate. Addition of [4-C-13]aspartate to the medium led to isotopic enrichment of several organic acids in the xylem sap. Quantitatively, malate carried the highest amount of label among the organic acids. Labelling of succinate indicates its formation by reversal of the TCA-cycle from oxaloacetate. Since aspartate was a prominent amino acid of the phloem sap, it is suggested that this is an important source of malate exported in the xylem. The export of these organic acids will play the role of removing electrons from the hypoxic roots, representing an additional mechanism in the metabolic response to root hypoxia. Malate, normally considered an intermediate in succinate formation, is definitively a product of anaerobic metabolism2848290CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP303931/2009-42013/03325-