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: PAG activity was stained for in a series of brain tissue sections with varying thicknesses to determine whether the thickness of the tissue section affects the detection of first-order reaction rates of PAG activity (Fig. 4). Figure 4 shows that 4-µm-thick sections were not reliable and 16-µm-thick sections were apparently too thick for the auxiliary GDH film to generate glutamate throughout the tissue sections. A thickness range of 6–12 µm showed similar PAG activity per µm tissue section thickness (Fig. 4B). This indicates that, in this tissue-thickness range, all glutamate produced by PAG was efficiently converted by the auxiliary GDH film, which resulted in reliable PAG staining in tissue sections ranging from 6–12 µm.
Affiliation: Department of Cell Biology and Histology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands (DB, WT, CJFVN).