Determination of glutamate dehydrogenase activity and its kinetics in mouse tissues using metabolic mapping (quantitative enzyme histochemistry).
Bottom Line: Glutamate dehydrogenase (GDH) catalyses the reversible conversion of glutamate into α-ketoglutarate with the concomitant reduction of NAD(P)(+) to NAD(P)H or vice versa.NAD(+)-dependent GDH V(max) was 2.5-fold higher than NADP(+)-dependent V(max), whereas the K(m) was similar, 1.92 mM versus 1.66 mM, when NAD(+) or NADP(+) was used, respectively.In all tissues, the highest activity was found when NAD(+) was used as a coenzyme.
Affiliation: Department of Cell Biology and Histology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands (DB, WT, CJFVN).Show MeSH
License 1 - License 2 - License 3
Mentions: To determine GDH kinetics, mouse liver tissue sections were metabolically mapped for GDH activity with eight different glutamate concentrations (0, 0.4, 1.4, 2, 5, 10, 30 and 50 mM) with either NAD+ (Fig. 3) or NADP+ (Fig. 4) as the coenzyme. The Km values of GDH for glutamate were similar when NAD+ or NADP+ was used as coenzyme (Fig. 5; Table 1), whereas the Vmax of GDH with NAD+ was 2.5-fold higher than with NADP+. Vmax and Km were optimal at 3 mM NAD+ or NADP+ (data not shown). GDH showed substrate inhibition both in the presence of NAD+ and NADP+ as the coenzyme. The dissociation constants (Ki) of this glutamate inhibition were 12.2 mM and 4.0 mM when NAD+ or NADP+ where used as the coenzyme, respectively. These Ki values indicate that GDH was less inhibited by its substrate glutamate when NAD+ was used as the coenzyme than when NADP+ was used, although this effect was not significant (Students t-test, p=0.29). Moreover, GDH activity was completely inhibited at higher glutamate concentrations when NADP+ was used as the coenzyme, but not when NAD+ was used as the coenzyme (Fig. 5). Glutamate did not affect the pH of the incubation medium. Therefore, the lower GDH activity at high glutamate levels was not caused by lower pH. Figure 5 shows that GDH Vmax was determined with a glutamate concentration of 10 mM in the incubation medium.
Affiliation: Department of Cell Biology and Histology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands (DB, WT, CJFVN).