Isolation, partial purification and characterization of isoenzymes of aspartate kinase from rice seeds

Abstract

Summary Aspartate kinase (AK, EC 2.7.2.4) and homoserine dehydrogenase (HSDH, EC 1.1.1.3) have been isolated and partially purified from immature rice (Oriza sativa L.) seeds. Three methods of protein separation (ammonium sulphate precipitation, anion-exchange chromatography and gel filtration chromatography) were used in order to purify and identify the isoenzymes of AK and HSDH. Two peaks of AK activity were eluted from the anion-exchange chromatography column (FFQ-Sepharose) with 230 and 286 mmol/L KCl. The first peak in elution order was inhibited by threonine, while the second peak was strongly inhibited by lysine. An optimum pH of 7.4 was determined for total AK activity. The optimum temperature for AK action was about 35 °C, whereas a combination of 12.5 mmol/L magnesium sulphate and 20 mmol/L ATP produced the highest level of activity. After the ammonium sulphate step, only one form of HSDH that was completely inhibited by threonine, was observed. The threonine-sensitive forms of HSDH and AK co-purified, independent of the protein purification method used exhibiting a molecular mass of 186 kDa. The lysine-sensitive AK had a molecular mass estimated to be 167 kDa. These results confirm the hypothesis that in higher plants, threonine and lysine-sensitive forms of AK are present and suggest that a bifunctional polypetide containing threonine-sensitive AK and HSDH is present is rice seeds.