Glutamate dehydrogenase from Medicago sativa L., purification and comparative kinetic studies of the organ-specific multiple forms

Abstract

NAD-specific glutamate dehydrogenase [L-glutamate: NAD+ oxidoreductase (deaminating) EC 1.4.1.2] from Medicago sativa constitutes organ-specific patterns of isoenzymes. The isoenzyme-patterns of seeds (GDH-I) and roots (GDH-II) were purified 1520-fold and 92-fold, respectively. All isoenzymes of both patterns remain stable throughout the purification procedures. Isoenzyme a7, the only isoenzyme common to both patterns was isolated from the GDH-I pattern. The three enzyme preparations were found to be identical in pH optima, substrate specificity and general kinetic properties. A comparative kinetic analysis revealed no pronounced differences between the various kinetic constants evaluated for the three enzyme preparations. Furthermore an identical order of substrate binding and product release could be established. Both initial rate measurements and product inhibition studies are consistent with an ordered ternary-binary kinetic mechanism. The results suggest that tissue-specific enzyme multiplicity of plant glutamate dehydrogenase is not related to differences in general or kinetic properties.