The pyridine nucleotide and non-pyridine nucleotide dependence of L-glutamate dehydrogenase in the histochemical system.

Abstract

Under histochemical conditions (fresh frozen sections from liver, kidney and cerebellum of the rat) it was shown that the oxidation of L-glutamic acid was carried out by the NAD-dependent L-glutamate dehydrogenase (E.C. 1.4.1.2) and/or the NAD- or NADP-dependent L-glutamate dehydrogenase (E.C. 1.4.1.3) as well as by an enzyme system which is not dependent on externally added NAD, NADP, FAD, FMN or CoQ10 for activity. This non-pyridine dependent activity was related to the L-glutamate dehydrogenases proper, owing to the following: a) the localization of activity noticed corresponds to that obtained with the NAD- or NADP-containing media, b) the incubation time needed for initial formation of red and blue formazans is similar to that with coenzyme-containing media, c) pre-extraction experiments reveal similarity in enzyme diffusion rates, d) the named activity is influenced by the same agents and to the same extent as the activity obtained by the inclusion of NAD or NADP (e.g. dissociation of the dehydrogenase molecule into subunits due to urea, inhibition of activity due to N-ethyl maleimide and 1.10-phenanthroline, activation due to the allosteric effect of ADP and to high substrate concentration, allosteric inhibition caused by GTP and inhibition caused by α-ketoglutaric acid, no inhibitory effect of KCN), and e) the named activity was not affected by added PMS (excluding activity due to L-aminoacid oxidase). In the in situ localization of enzyme activity it was found that L-glutamate dehydrogenases E.C. 1.4.1.2 and E.C. 1.4.1.3 co-exist in the cells of kidney and cerebellum, while the L-glutamate dehydrogenase E.C. 1.4.1.3 only was present in liver cells. Finally, it was stated that incubation time should be kept as short as possible in order to avoid “Nothing dehydrogenase” reaction as well as inhibition due to accumulation of α-ketoglutaric acid. Only “gel” incubation media should be applied.