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Determination of phosphate-activated glutaminase activity and its kinetics in mouse tissues using metabolic mapping (quantitative enzyme histochemistry).

Botman D, Tigchelaar W, Van Noorden CJ - J. Histochem. Cytochem. (2014)

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Histology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands (DB, WT, CJFVN).

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Principle of metabolic mapping of phosphate-activated glutaminase (PAG) using a tetrazolium salt as final electron acceptor. By adding a surplus of glutamate dehydrogenase (GDH) underneath the tissue sections it is possible to stain PAG activity because GDH uses NAD(P)+ as coenzyme. Abbreviations: (m)PMS, (methoxy)phenazine methosulfate; NitroBT, nitrotetrazolium blue chloride.
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fig2-0022155414551177: Principle of metabolic mapping of phosphate-activated glutaminase (PAG) using a tetrazolium salt as final electron acceptor. By adding a surplus of glutamate dehydrogenase (GDH) underneath the tissue sections it is possible to stain PAG activity because GDH uses NAD(P)+ as coenzyme. Abbreviations: (m)PMS, (methoxy)phenazine methosulfate; NitroBT, nitrotetrazolium blue chloride.

Mentions: A method for metabolic mapping of PAG activity is currently not available. Therefore, we developed a novel method with GDH as an auxiliary enzyme using a two-step reaction (Fig. 2; Botman et al. In Press; Van Noorden and Frederiks 1992). Metabolic mapping with a two-step reaction has been used before for other enzymes that also do not use NAD+ or NADP+ as coenzyme to visualize their activity by using the activity of an enzyme that can convert a tetrazolium salt into formazan (Van Noorden and Frederiks 1992).


Determination of phosphate-activated glutaminase activity and its kinetics in mouse tissues using metabolic mapping (quantitative enzyme histochemistry).

Botman D, Tigchelaar W, Van Noorden CJ - J. Histochem. Cytochem. (2014)

Principle of metabolic mapping of phosphate-activated glutaminase (PAG) using a tetrazolium salt as final electron acceptor. By adding a surplus of glutamate dehydrogenase (GDH) underneath the tissue sections it is possible to stain PAG activity because GDH uses NAD(P)+ as coenzyme. Abbreviations: (m)PMS, (methoxy)phenazine methosulfate; NitroBT, nitrotetrazolium blue chloride.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2 - License 3
Show All Figures
getmorefigures.php?uid=PMC4230542&req=5

fig2-0022155414551177: Principle of metabolic mapping of phosphate-activated glutaminase (PAG) using a tetrazolium salt as final electron acceptor. By adding a surplus of glutamate dehydrogenase (GDH) underneath the tissue sections it is possible to stain PAG activity because GDH uses NAD(P)+ as coenzyme. Abbreviations: (m)PMS, (methoxy)phenazine methosulfate; NitroBT, nitrotetrazolium blue chloride.
Mentions: A method for metabolic mapping of PAG activity is currently not available. Therefore, we developed a novel method with GDH as an auxiliary enzyme using a two-step reaction (Fig. 2; Botman et al. In Press; Van Noorden and Frederiks 1992). Metabolic mapping with a two-step reaction has been used before for other enzymes that also do not use NAD+ or NADP+ as coenzyme to visualize their activity by using the activity of an enzyme that can convert a tetrazolium salt into formazan (Van Noorden and Frederiks 1992).

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Histology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands (DB, WT, CJFVN).

Show MeSH