Purification and properties of glutamate dehydrogenase from the cold-hardy gall fly larva, Eurosta solidaginis
Glutamate dehydrogenase (GDH) from the goldenrod gall fly larva, Eurosta solidaginis, was purified 500-fold to a final specific activity of 37.5 μmol NADH utilized/min mg-1 protein. The enzyme is a hexamer of mol. wt 350,000 ± 30,000 and subunit size 57,000 ± 5000. Both nicotinamide coenzymes were utilized with activity ratios, NADH/NADPH, of 3.5 at pH 6.75 and 11 at pH 7.5; NAD/NADP activity ratios were 7 at pH 7.5 and 15 at pH 8. Enzyme kinetic constants (S0.5) for NH4 +, α-ketoglutarate and coenzyme were 210 ± 10, 2.7 ± 0.2 and 0.032 ± 0.001 mM for the NADH linked reaction and 61 ± 2.0, 0.56 ± 0.06 and 0.060 ± 0.003 mM for the NADPH reaction. Ammonium ion kinetics were distinctly sigmoidal. Metabolite effectors modified NH4 + kinetics, ADP activated, decreasing S0.5 for NH4 + four-fold and reducing n, the Hill coefficient. ATP inhibited the NADH linked reaction but activated the NADPH reaction. GTP inhibited the enzyme. GDH activity in the forward, glutamate oxidizing, direction was undetectable in the absence of ADP. S0.5 values for glutamate and coenzyme (at saturating ADP) were 3.1 ± 0.2 and 0.22 ± 0.02 mM for the NAD reaction and 3.8 ± 0.3 and 0.20 ± 0.02 mM for the NADP reaction. Inhibitors (GTP, ATP) modified both glutamate and coenzyme kinetics. Arrhenius plots were linear over the range 25-0°C and temperature had little effect on enzyme substrate affinity. Inhibitor effects, however, were altered with temperature; I50 values for GTP and ATP decreased at low temperature and ADP reversal of GTP inhibition of the NADPH linked reaction was greater at 5°C than at 25°C. Larval GDH appears well suited for a role in amino acid biosynthesis and may have an important role in the overwintering accumulation of proline in this freezing-tolerant insect.
|Keywords||cold hardiness, enzyme regulation, Eurosta solidaginis, glutamate dehydrogenase, larval enzyme kinetics, low temperature enzyme function|
Male, K.B. (Keith B.), & Storey, K. (1982). Purification and properties of glutamate dehydrogenase from the cold-hardy gall fly larva, Eurosta solidaginis. Insect Biochemistry, 12(5), 507–514. doi:10.1016/0020-1790(82)90019-1