Characterization of Aspartate Aminotransferase Isoenzymes from Leaves of Lupinus albus L. cv Estoril

다국어 초록 (Multilingual Abstract)

Two aspartate azninotransferase (EC 2.6.1.1) isoenzymes (AAT-1 and AAT-2) from Lupines albus L. cv Estoril were separated, purified, and characterized. The molecular weight, pI value, optimum pH, optimum temperature, and thermodynamic parameters for thermal inactivation of both isaenzymes were abtained. Studies of the kinetic mechanism, and the kinetics of product inhibition and high substrate concentration inhibition, were performed. The effect of some divalent ions and irreversible inhibitors on both AAT isoenzymes was also studied. Native PAGE showed a higher molecular weight for AAT 2 compared with AAT 1. AAT 1 appears to be more anionic than AAT 2, which was suggested by the anion exchange chromatography SDS-PAGE showed a similar sub-unit molecular weight for both isoenzymes. The optimum pH (between 8.0 and 9.0) and temperature (60-65℃) were similar far both isoenzymes. In the temperature range of 45-65℃, AAT-2 has higher thermostability than AAT-1. Both isoenzymes showed a high affinity for keto-acid substrates, as well as a higher affinity to aspartate than glutamate. Manganese ions induced an increase in both AAT isoenzymes activities, but no cooperative effect was detected. Among the inhibitors tested, hydroxylamine affected both isoenzymes activity by an irreversible inhibition mechanism.