Proteins (YGL202W, YJL060W, YER152C, YDL168W) were expressed from Open Biosystems Yeast ORF clones (http://www.openbiosystems.com/GeneExpression/Yeast/ORF/)[1]. Following growth and galactose induction, bacteria were stored as frozen cell pellets at -80 °C. After disruption of the frozen cells with zirconium-silica beads, the polyhistidine-tagged proteins were purified using Ni-NTA superflow cartridges (Qiagen) and verified by SDS PAGE.
Activity was determined in the direction of glutamate formation essentially as previously described [2]. Purified protein (5 µl) was incubated with L-2- aminoadipate (1.25 mM), yeast glutamate dehydrogenase (10U - Sigma 49392), NADP (0.2 mM), pyridoxal phosphate (1 mg/ml) in 20 mM Tris/HCl buffer, pH8.0. After mixing and pre-equilibrating at 37 °C for 5 min, the reaction was started by adding 2-oxo-glutarate (2.5 mM) to give a final volume of 3 ml. The increase in A340nm due to NADPH production was monitored with time (glutamate produced by transamination is deaminated by the glutamate dehydrogenase, producing NADPH & ammonia and regenerating 2-oxoglutarate).
[1] Gelperin DM, White MA, Wilkinson ML, Kon Y, Kung LA, Wise KJ, Lopez- Hoyo N, Jiang L, Piccirillo S, Yu H, Gerstein M, Dumont ME, Phizicky, EM, Snyder M, Grayhack EJ (2005). Biochemical and genetic analysis of the yeast proteome with a movable ORF collection. Genes and Development 19, 2816-2826.
[2] Young M. (1973) Studies on the growth in culture of plant cells. XVI. Nitrogen assimilation during nitrogen-limited growth of Acer pseudoplatanus L. cells in chemostat culture. Journal of Experimental Botany 24, 1172-1185.