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: We used tetrazolium salts for the metabolic mapping of the activity of dehydrogenases (Fig. 2). In this method, the dehydrogenase, in this case GDH, reduces NAD(P)+ to NAD(P)H. NAD(P)H reduces an electron carrier that is present in the medium and subsequently reduces the water-soluble slightly yellow nitro blue tetrazolium (NitroBT) into a water-insoluble blue formazan precipitate. The absorbance of the precipitated formazan at the site of GDH activity is therefore a direct measure of GDH activity (Chieco et al. 2013; Van Noorden 2010; Jonker et al. 1996; Van Noorden and Frederiks 1992). This methodology enables the assessment of GDH activity in its intact cellular microenvironment when unfixed cryostat sections are used. Chemical fixation affects (usually inhibits) enzyme activity. For proper localization of the enzyme activity by the generated formazan, macromolecules have to be kept in the tissue section during enzyme incubation (Van Noorden 2010; Van Noorden and Vogels 1989). One of the best methods to achieve this is addition of the water-soluble polymer polyvinyl alcohol (PVA) to the incubation medium. In PVA-containing media, small molecules, such as substrates and coenzymes, can diffuse freely, but large molecules, such as proteins, cannot. Additionally, PVA keeps the tissue morphology intact. This methodology ensures posttranslational modifications to the enzyme and its microenvironment are kept intact as much as possible.
Affiliation: Department of Cell Biology and Histology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands (DB, WT, CJFVN).