Bovine glutamate dehydrogenase

The K_m was determined using a modified coupled assay (Hawkes et al., 1995 (link)). Assay buffer was combined with bovine glutamate dehydrogenase (Sigma) and a large excess of imidazoleacetol-phosphate transaminase (S. Singh, Syngenta). Reduced nicotinamide adenine dinucleotide (Sigma) and IGPD2 were added to a final concentration of 6 mM and 45 nM, respectively, before incubation at 30°C in a temperature-controlled spectrophotometer. When the signal at 340 nm had stabilized, IGP was added at various concentrations to start the reaction. Initial rates from the linear part of the curve were recorded, and K_m and K_cat were determined by non-linear least-squares fitting in GraphPad Prism.

Glutaminase activity was measured as described previously [17 (link)]. Samples of 8 μM PdxS, 8 μM PdxT, or 8 μM mixture of both proteins was incubated with 10 mM glutamine, 6 units of bovine glutamate dehydrogenase, and 0.5 mM 3-acetylpyridine adenine dinucleotide (APAD; Sigma) in a total reaction volume of 300 μl of 50 mM Tris-Cl (pH 8.0). The reaction mixture was incubated at 37°C. The absorbance of each sample was read at a wavelength of 363 nm (OD₃₆₃).