Isolamento, purificação parcial e caracterização bioquimica da aspartato quinase e homoserina desidrogenase de sementes de sorgo

AUTOR(ES)
DATA DE PUBLICAÇÃO

2004

RESUMO

Cereal seeds are poor sources of essential amino acids, in particular lysine and threonine, which are synthesized as part of the aspartic acid metabolic pathway. Many of the enzymes of this pathway have been studied in several higher plants species, however, the metabolism of lysine and threonine in sorghum is unknown. In this work were studied two of the enzymes of this pathway in sorghum, aspartate kinase (AK) and homoserine dehydrogenates (HSDH). Assay conditions were optimized for the determination of AK and HSDH activities. The highest levels of activity for both enzymes were observed from immature sorghum seeds using 50 J.1L of sample and an incubation period from 10 to 30 min. The co-factor that had the greatest effect on HSDH activity was NADP. To separate, purify and identify the various isoforms of the AK and HSDH from sorghum seeds, the extracts were subjected to ammonium sulphate precipitation, anionexchange chromatography and gel filtration chromatography. Two AK activity peaks at 183 and 262 mmol.L-1 KCL were eluted by anion-exchange chromatography (DEAE-Sephacel). Both of these activity peaks were strongly inhibited by lysine. For HSDH, two activity peaks were eluded at 145 and 183 mmol.L"1 KCI, with the first peak being resistant to threonine inhibition while the second was sensitive to threonine inhibition. However, through gel filtration chromatography (Sephacryl S-200), one peak of AK activity co-eluted with one peak of HSDH and both were sensitive to threonine inhibition, suggesting the presence of a bifunctional AKHSDH isoenzyme in sorghum, with a molecular mass estimated as 164 kDa. Using these protein separation methods, AK was partially purified by 30-fold when compared to the crude extract. The activities of AK and HSDH were studied in the presence of lysine, threonine, methionine, valine, calcium, EGTA, calmodulin, SAM, AEC and increasing concentrations of KCl. AK showed inhibition by threonine and lysine confirming the existence of two isoenzymes, one sensitive to threonine and the other sensitive to lysine, the latter being predominant in sorghum seeds. Methionine, SAM plus lysine and AEC also inhibited AK activity, however, increasing KCI concentrations and calcium did not produce any significant effect on AK activity, confirming that calcium does not play a role in AK regulation in sorghum seeds. HSDH also exhibited some inhibition by threonine, but the majority of its activity was not inhibited, thus indicating the existence of a threonine sensitive isoenzyme and a second predominant isoenzyme, which was resistant to threonine inhibition. Valine and SAM plus threonine also inhibited HSDH, however, increasing concentrations of KCl and calcium had no inhibitory effect. Regarding seed storage proteins of sorghum, the commercial hibrid MASSA 03 used in this study showed a high concentration of glutelins and a low concentration of kafirins similarly to what was described for several high lysine mutants lines. Furthermore, the concentration of soluble amino acids was also compatible to those of the high lysine mutant lines, which suggest that this commercial hibrid may also be considered as a high lysine sorghum. Finally, for genetic improvement programs, the sorghum mutant genotype 1S 16227 should be evaluated due to its higher contents and quality of its seed storage proteins and soluble amino acid

ASSUNTO(S)

sorgo

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