title: Determination of glutamate dehydrogenase activity and its kinetics in mouse tissues using metabolic mapping (quantitative enzyme histochemistry).
abstract: 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. GDH activity is subject to complex allosteric regulation including substrate inhibition. To determine GDH kinetics in situ, we assessed the effects of various glutamate concentrations in combination with either the coenzyme NAD(+) or NADP(+) on GDH activity in mouse liver cryostat sections using metabolic mapping. 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. With either coenzyme, V(max) was determined at 10 mM glutamate and substrate inhibition was observed at higher glutamate concentrations with a K(i) of 12.2 and 3.95 for NAD(+) and NADP(+) used as coenzyme, respectively. NAD(+)- and NADP(+)-dependent GDH activities were examined in various mouse tissues. GDH activity was highest in liver and much lower in other tissues. In all tissues, the highest activity was found when NAD(+) was used as a coenzyme. In conclusion, GDH activity in mice is highest in the liver with NAD(+) as a coenzyme and highest GDH activity was determined at a glutamate concentration of 10 mM.
authors.affiliation: Department of Cell Biology and Histology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands (DB, WT, CJFVN).
authors.forename: Dennis
authors.initials: D
authors.lastname: Botman
authors.affiliation: Department of Cell Biology and Histology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands (DB, WT, CJFVN).
authors.forename: Wikky
authors.initials: W
authors.lastname: Tigchelaar
authors.affiliation: Department of Cell Biology and Histology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands (DB, WT, CJFVN) c.j.vannoorden@amc.uva.nl.
authors.forename: Cornelis J F
authors.initials: CJ
authors.lastname: Van Noorden
mesh_terms: D000818:Animals; D004789:Enzyme Activation; D005969:Glutamate Dehydrogenase / Q000378:metabolism*; D019044:Image Cytometry; D007700:Kinetics; D008099:Liver / Q000201:enzymology* / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL
chemical_list: D005969:Glutamate Dehydrogenase
keywords: enzyme cytochemistry; enzyme inhibition; enzymes; glutamate; quantitation