The isozymic nature and kinetic properties of glutamate dehydrogenase from safflower seedlings

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Levels of glutamate dehydrogenase (GDH) [L-glutamate : NAD oxidoreductase (deaminating), EC 1.4.1.2] from safflower roots and cotyledons increased (×2.7) and decreased (× 5.7), respectively, as a function of seedling age. No significant changes in enzyme levels were detected during hypocotyl development. GDH preparations of the different organs were resolved by polyacrylamide gel electrophoresis into 2 to 4 isozymes. The isozymic pattern was influenced by seedling age and organ tested. The slowest moving isozyme (No. 1 ) appears to be responsible for the changes in GDH levels observed in cotyledons and roots. We isolated isozyme 1 and a GDH fraction chiefly containing isozyme 2, by DEAE-cellulose chromatography. GDH was purified approximately 53-fold from the particulate fraction of cotyledons. The pH optima for NADH and NAD activities were 8.2 and 8.9, respectively. Michaelis constants were found to be: a-ketoglutarate, 8 mM; glutamate, 4 mM; ammonium, 35.4 mM; NAD, 0.26 mM; NADH, 0.065 mM. Km values of isozymes 1 and 2 were similar. The binding order of substrates in the reductive amination reaction was NADH, a-ketoglutarate and NH_4^+.
日本植物生理学会の論文