Determination of aspartate kinase activity in maize tissues

Lysine, threonine, methionine and isoleucine are synthesized from aspartate in a branched pathway in higher plants. Aspartate kinase plays a key role in the control of the aspartate pathway. The enzyme is very sensitive to manipulation and storage and the hydroxamate assay normally used to determine aspartate kinase activity has to be altered according to the plant species and tissue to be analyzed. We have optimized the assay for the determination of aspartate kinase in maize plants callus cell cultures. Among all the assay parameters tested, the concentration of ATP/Mg and temperature were critical for enzyme activity. In the case of temperature, 35°C was shown to be the optimum temperature for aspartate kinase activity.Lisina, treonina, metionina e isoleucina são derivados do aspartato. Aspartato quinase tem um papel chave no controle da via metabólica do aspartato. A enzima é muito sensível à manipulação e armazenagem e o ensaio enzimático do hidroxamato que é normalmente utilizado para determinar a atividade da aspartato quinase deve ser alterado de acordo com a espécie de planta e tecido vegetal a ser analisado. Nós otimizamos o ensaio para a determinação da atividade da aspartato quinase em culturas celulares de calos de milho. Entre os vários parâmetros testados do ensaio, a concentração de ATP/Mg e temperatura foram críticos para atividade enzimática. No caso da temperatura, 35°C foi a temperatura ótima para atividade da aspartato quinase

Nutritional Quality of Sorghum Seeds: Storage Proteins and Amino Acids

One sorghum commercial genotype (MASSA 03) and nine ICRISAT high-lysine genotypes from India were analyzed for storage protein content, distribution profile, and soluble amino acid concentrations. Storage proteins fraction were extracted and separated by SDS-PAGE. Soluble amino acids contents were determined by HPLC. Variations in intensity and appearance and disappearance of protein bands were observed among the sorghum genotypes suggesting genetic variability. Amino acid profile also indicated large variations in the amino acid concentrations. The high lysine and threonine soluble concentrations observed in the seeds of the sorghum genotypes encouraged the use of these genotypes as potential food source due to the better balanced amino acids profile.FAPESP Fundacao de Amparo a Pesquisa do Estado de Sao Paulo, Brazil[04/16039-4]Coordena ao de Aperfei oamento de Pessoal de N vel Superior (CAPES, Brazil)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq, Brazil

Isolamento de enzimas envolvidas na biossíntese de treonina em sementes de sorgo

Cereal seeds are poor in essential amino acids, particularly lysine, tryptophan and threonine. The amino acids lysine and threonine are synthesized in the aspartate pathway. Although most of the enzymes of the aspartate pathway have been isolated and characterized in higher plant species, the metabolism of lysine and threonine is totally unknown in sorghum. We have isolated two enzymes, aspartate kinase (AK) and homoserine dehydrogenase (HSDH) from sorghum. Optimum assay conditions were established for the determination of AK and HSDH activities. The highest level of activity was observed in immature seeds. AK was shown to be inhibited by threonine and lysine indicating the existence of at least two isoenzymes, one sensitive to threonine inhibition and the other sensitive to lysine inhibition with the latter being predominant in sorghum seeds. HSDH was shown to be inhibited by threonine indicating the existence of a threonine-sensitive HSDH, however, most of the activity was not inhibited by threonine, suggesting the existence of a second predominant isoenzyme of HSDH resistant to threonine inhibition.As sementes dos cereais são pobres em aminoácidos essenciais, principalmente lisina, triptofano e treonina. Os aminoácidos lisina e treonina são sintetizados na via metabólica do aspartato. Apesar de a maioria das enzimas da via do aspartato ter sido isolada e caracterizada em várias espécies de plantas, o metabolismo de lisina e treonina é totalmente desconhecido em sorgo. Isolamos duas enzimas, aspartato quinase (AK) e homosserina desidrogenase (HSDH) de sorgo, estabelecendo condições ótimas de ensaio para determinação da atividade de AK e HSDH. Observaram-se as atividades mais elevadas de AK e HSDH em sementes imaturas. A atividade de AK foi inibida por treonina e lisina, indicando a existência de duas isoenzimas, uma sensível à inibição por treonina e, a outra, sensível à inibição pela lisina, predominando, esta última, nas sementes de sorgo. A atividade de HSDH foi inibida por treonina, indicando a existência de uma isoenzima sensível à treonina, a maioria da atividade, entretanto, não foi inibida por treonina, sugerindo a existência de uma segunda isoenzima de HSDH predominante resistente à inibição pela treonina.95104Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Determination of aspartate kinase activity in maize tissues

Lysine, threonine, methionine and isoleucine are synthesized from aspartate in a branched pathway in higher plants. Aspartate kinase plays a key role in the control of the aspartate pathway. The enzyme is very sensitive to manipulation and storage and the hydroxamate assay normally used to determine aspartate kinase activity has to be altered according to the plant species and tissue to be analyzed. We have optimized the assay for the determination of aspartate kinase in maize plants callus cell cultures. Among all the assay parameters tested, the concentration of ATP/Mg and temperature were critical for enzyme activity. In the case of temperature, 35degreesC was shown to be the optimum temperature for aspartate kinase activity

Saccharopine Dehydrogenase Activity in the High-Lysine Opaque and Floury Maize Mutants.

Lysine is an essential amino acid normally present in very low concentration in cereal seeds. In previous reports we have studied the metabolism of lysine in several distinct high-lysine maize mutants and observed drastic variations in the activity of saccharopine dehydrogenase (SDH), a key enzyme involved in lysine degradation. We have now analyzed the activity of SDH using non-denaturing polyacrylamide gel electrophoresis (PAGE) to identify possible isoenzymes that could explain the patterns of activity previously observed. The results indicated the presence of at least two SDH isoenzymes, one contributing to approximately 90% of the total enzyme activity and a minor form only present in the wild type lines and the opaque-1 mutant. The results suggest that the differences in total SDH activity among the genotypes tested are due to alterations in the predominant SDH isoenzymic form, which is likely to be the bifunctional polypeptide containing lysine 2-oxoglutarate reductase

Determination of aspartate kinase in maize tissues.

Lysine, threonine, methionine and isoleucine are synthesized from aspartate in a branched pathway in higher plants. Aspartate kinase plays a key role in the control of the aspartate pathway. The enzyme is very sensitive to manipulation and storage and the hydroxamate assay normally used to determine aspartate kinase activity has to be altered according to the plant species and tissue to be analyzed. We have optimized the assay for the determination of aspartate kinase in maize plants callus cell cultures. Among all the assay parameters tested, the concentration of ATP/Mg and temperature were critical for enzyme activity. In the case of temperature, 35°C was shown to be the optimum temperature for aspartate kinase activity