The use of ion exchange chromatography for the production of homogeneous glutamate dehydrogenase from wheat seedlings

Abstract

Homogeneous glutamate dehydrogenase (GDH, L-glutamate: NAD (P) H-oxidoreductase 1.4.1.3), purified 319.5 times with a specific activity of 377 U/mg protein and a yield of 3.7 % was obtained using 5-stages purification. Usually, the purification of enzymes in our laboratory is carried out according to the following general scheme: homogenization of plant material (obtaining an enzyme extract), fractionation of enzyme proteins with ammonium sulphate, gel filtration on Sephadex G-25, providing desalination of proteins, ion exchange chromatography on DEAE-Sephacel, desorption of enzymatic activity was carried out with a NaCl concentration gradient (0.15-0.3 M). The last stage of purification was carried out using gel chromatography on a Sephadex G-200. It is known that ATP and metabolic intermediates (glutamate and 2-oxoglutarate) have an inhibitory effect on glutamate dehydrogenase. The activation of enzymatic activity was observed under the action of AMP, calcium and magnesium ions, and ammonium sulphate. The use of DEAE-Sephacel had the major effect out of all chromatographic methods (ion exchange chromatography, partition chromatography, gel filtration, etc.). In this case, the degree of GDH purification was more than 300 times.

The performed PAGE-electrophoresis, followed by specific staining using tetrazolium method, revealed glutamate dehydrogenase activity in the purified protein. The pH optimum of the obtained GDH for the reductive amination and oxidative deamination reaction was 8.5. Kinetic characteristics of the affinity of glutamate dehydrogenase to various substrates were obtained. The Michaelis constant for 2-oxoglutarate was 2.75, and for glutamate it was 14.9, which may indicate the predominance of β-subunits in the structure of the GDH oligomer.  Analysis of these data indicates a significantly higher affinity of the studied enzyme for 2-oxoglutarate.