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
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Mentions: GDH is a key enzyme in mitochondrial carbohydrate metabolism and catalyses the final step in glutaminolysis after the conversion of glutamine to glutamate by phosphate-activated glutaminase (PAG; Fig. 1). Via this pathway, α-KG can be shuttled into the tricarboxylic acid (TCA) cycle (DeBerardinis et al. 2008, 2007; Reitzer et al. 1979). This process is called anaplerosis, which can generate ATP by oxidation of α-KG, and raises the ATP:ADP ratio to induce insulin secretion (Treberg et al. 2010; Carobbio et al. 2009; Stanley et al. 2000, 1998). Alternatively, α-KG can be converted into citrate by IDH1 and IDH2, and aconitase for lipid synthesis via the formation of acetyl coenzyme A (AcCoA) (Brose et al. 2013; Filipp et al. 2012; Metallo et al. 2012; Collins et al. 2011; Mullen et al. 2011; DeBerardinis et al. 2007). On the other hand, glutamate can also be converted into malate via the TCA cycle or into pyruvate by malic enzyme, generating NADPH required for cellular stress protection and for lipid and cholesterol metabolism (Koehler and Van Noorden 2003; Koh et al. 2004; DeBerardinis et al. 2007; Wise et al. 2008; Romero-Garcia et al. 2011; Lorin et al. 2013). Additionally, GDH activation by leucine facilitates the conversion of GDP to GTP, resulting in the activation of the small GTPase Rag, which activates mammalian target of rapamycin complex (mTORC), and results in cell growth and decreased autophagy (Lorin et al. 2013; Durán et al. 2012). Next to cellular carbohydrate metabolism, GDH is involved in ureagenesis, as the conversion of glutamate into α-KG also produces NH3+, which can be used to produce carbamoyl phosphate for the urea cycle (Spanaki and Plaitakis 2012; Treberg et al. 2010; Stanley et al. 2000, 1998; Boon et al. 1999; Nissim et al. 1992). Finally, GDH activity is increased in various brain pathologies, such as Parkinson’s disease, schizophrenia and brain cancer (Plaitakis et al. 2010; Bao et al. 2009; Yang et al. 2009; Burbaeva et al. 2003). It is hypothesised that the increased GDH activity alters neuronal cell metabolism and increases the production of reactive oxygen species (ROS) through glutamate oxidation, which can damage neuronal cells (Plaitakis et al. 2010).
Affiliation: Department of Cell Biology and Histology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands (DB, WT, CJFVN).