Characterization of aspartate aminotransferase isoenzymes from leaves of Lupinus albus L. cv Estoril.
Two aspartate aminotransferase (EC 2.6.1.1) isoenzymes (AAT-1 and AAT-2) from Lupinus 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 isoenzymes were obtained. 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 degrees C) were similar for both isoenzymes. In the temperature range of 45-65 degrees C, 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.
Martins
Maria Luisa Louro
ML
Instituto Superior de Agronomia, Departamento de Quimica Agricola e Ambiental, Tapada da Ajuda, 1349-017 Lisboa, Portugal. luisalouro@isa.utl.pt
Mourato
Miguel Pedro de Freitas Barbosa
MP
de Varennes e Mendonça
Amarilis Paula Alberti
AP
D001219:Aspartate Aminotransferases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002413:Cations, Divalent / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D019811:Hydroxylamine / Q000494:pharmacology; D007526:Isoelectric Point; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D031297:Lupinus / Q000201:enzymology*; D008970:Molecular Weight; D018515:Plant Leaves / Q000201:enzymology; D013379:Substrate Specificity; D013816:Thermodynamics
D002413:Cations, Divalent; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D019811:Hydroxylamine; D001219:Aspartate Aminotransferases