Determination of phosphate-activated glutaminase activity and its kinetics in mouse tissues using metabolic mapping (quantitative enzyme histochemistry).
Bottom Line: PAG activity was decreased to 22% in the presence of 2 mM 6-diazo-5-oxo-L-norleucine.When compared with liver, kidney and brain, other tissues showed 3-fold to 6-fold less PAG activity.In conclusion, PAG is mainly active in mouse kidney, brain and liver, and shows different kinetics depending on which type of PAG is expressed.
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: Phosphate-activated glutaminase (PAG, EC 184.108.40.206) catalyzes the conversion of glutamine to glutamate as is shown in Fig. 1 (Curthoys 1995; Botman et al. In Press). PAG is considered to be a mitochondrial enzyme, although PAG activity has also been found in nuclei (Campos-Sandoval et al. 2007; Olalla et al. 2002). The conversion of glutamine to glutamate by PAG is the first step in glutaminolysis (Plaitakis et al. 2011; Mastorodemos et al. 2009, 2005). The second step is the conversion of glutamate to α-ketoglutarate (α-KG) by glutamate dehydrogenase (GDH). Glutaminolysis has emerged as a potential therapeutic target in brain tumors (van Lith et al. 2014; van Lith et al. In Press; Mohrenz et al. 2013; Seltzer et al. 2010). In particular, secondary glioblastoma, which possesses an isocitrate dehydrogenase (IDH) 1 or 2 mutation (Balss et al. 2008; Parsons et al. 2008; Ichimura et al. 2009; Nobusawa et al. 2009; Sanson et al. 2009; Yan et al. 2009; Bleeker et al. 2010, 2008), is considered to be sensitive to glutaminolysis inhibition because its product, α-KG, is the substrate for mutated IDH1 and IDH2. To enable metabolic mapping of glutaminolysis, we developed and optimized methods for the metabolic mapping of GDH (Botman et al. In Press) and PAG; the latter is described here.
Affiliation: Department of Cell Biology and Histology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands (DB, WT, CJFVN).