Determination of Glutamate Dehydrogenase Activity and Its Kinetics in Mouse Tissues using Metabolic Mapping (Quantitative Enzyme Histochemistry)

作者：Botman Dennis; Tigchelaar Wikky; Van Noorden Cornelis J F^*

来源：Journal of Histochemistry and Cytochemistry, 2014, 62(11): 802-812.

DOI：10.1369/0022155414549071

• glutamate
• enzyme cytochemistry
• enzymes
• enzyme inhibition
• quantitation

Glutamate dehydrogenase (GDH) catalyses the reversible conversion of glutamate into -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.

• 出版日期2014-11