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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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(A) Metabolic mapping of phosphate-activated glutaminase (PAG) activity in the presence of 37.5 mM glutamine in tissue sections from mouse brain of various thicknesses. (B) Absorbance, as calculated per µm tissue section thickness. All test reactions were corrected for nonspecific background staining in the absence of substrate. Absorbance is presented as the mean absorbance ± SEM (n=8 measurements for 4, 6, 10 and 16 µm and n=9 measurements for 8 and 12 µm).
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fig4-0022155414551177: (A) Metabolic mapping of phosphate-activated glutaminase (PAG) activity in the presence of 37.5 mM glutamine in tissue sections from mouse brain of various thicknesses. (B) Absorbance, as calculated per µm tissue section thickness. All test reactions were corrected for nonspecific background staining in the absence of substrate. Absorbance is presented as the mean absorbance ± SEM (n=8 measurements for 4, 6, 10 and 16 µm and n=9 measurements for 8 and 12 µm).

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.


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)

(A) Metabolic mapping of phosphate-activated glutaminase (PAG) activity in the presence of 37.5 mM glutamine in tissue sections from mouse brain of various thicknesses. (B) Absorbance, as calculated per µm tissue section thickness. All test reactions were corrected for nonspecific background staining in the absence of substrate. Absorbance is presented as the mean absorbance ± SEM (n=8 measurements for 4, 6, 10 and 16 µm and n=9 measurements for 8 and 12 µm).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig4-0022155414551177: (A) Metabolic mapping of phosphate-activated glutaminase (PAG) activity in the presence of 37.5 mM glutamine in tissue sections from mouse brain of various thicknesses. (B) Absorbance, as calculated per µm tissue section thickness. All test reactions were corrected for nonspecific background staining in the absence of substrate. Absorbance is presented as the mean absorbance ± SEM (n=8 measurements for 4, 6, 10 and 16 µm and n=9 measurements for 8 and 12 µm).
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.

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
Related in: MedlinePlus