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 M. 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 3 enzyme preparations were 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 3 enzyme preparations. An identical order of substrate binding and product release was established. Both initial rate measurements and product inhibition studies are consistent with an ordered ternary-binary kinetic mechanism. Tissue-specific enzyme multiplicity of plant glutamate dehydrogenase is probably not related to differences in general or kinetic properties.