MALATE - A POSSIBLE SOURCE OF ERROR IN THE NAD GLUTAMATE-DEHYDROGENASE ASSAY

The effects of externally induced metabolic perturbations are often studied through changes of the enzyme activity patterns in crude plant extracts. From glutamate dehydrogenase (GDH) it is reported that environmental changes not only influence the amount of the enzymatic activity, but also the ratio of the aminating to the deaminating activities (NADH/NAD+ ratio). Using crude cell extracts of suspension cultures of wheat (Triticum aestivum L. cv. Heines Koga II) we find evidence that the pretreatment of the homogenate directly influences this ratio. Dialysis of these crude cell extracts resulted in a 70% loss of the NAD+ activity, while the NADH activity remained unchanged. The deaminating activity in the dialysed extract could be completely restored upon addition of a dialysable factor which was identified to be malate. The interference of malate with the glutamate dehydrogenase reaction is caused through the action of malate dehydrogenase and glutamate oxaloacetate transaminase which are both present in high activities in the extracts. Only in exhaustively dialysed cell extracts can the proper deaminating GDH activity be determined. The results are discussed in the light of the controversial reports on the variable ratio of the NADH/NAD+ activity of GDH.