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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) Box-and-whisker plot (Tukey test) of formazan absorbance as a measure of the activity of glutamate dehydrogenase (GDH) films on glass slides. GDH was evenly distributed over the glass slides (n=10 measurements for films 1 and 3, n=11 measurements for film 2). Dot in film 2 data respresents outlier. (B) Phosphate-activated glutaminase (PAG) activity in liver, brain and kidney sections in either the presence or absence of an auxiliary GDH film underneath the cryostat tissue sections. All absorbance measurements were corrected for nonspecific background staining in the absence of substrate. Activity is presented as the mean reaction velocity ± SEM (n=3).
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fig3-0022155414551177: (A) Box-and-whisker plot (Tukey test) of formazan absorbance as a measure of the activity of glutamate dehydrogenase (GDH) films on glass slides. GDH was evenly distributed over the glass slides (n=10 measurements for films 1 and 3, n=11 measurements for film 2). Dot in film 2 data respresents outlier. (B) Phosphate-activated glutaminase (PAG) activity in liver, brain and kidney sections in either the presence or absence of an auxiliary GDH film underneath the cryostat tissue sections. All absorbance measurements were corrected for nonspecific background staining in the absence of substrate. Activity is presented as the mean reaction velocity ± SEM (n=3).

Mentions: To ascertain whether the auxiliary GDH activity of the GDH films between cryostat sections and glass slides were available throughout the tissue sections in a zero order manner (i.e., not rate limiting), we determined the distribution of GDH activity over the glass slides (Fig. 3A). GDH activity on the glass slides showed little variation over the entire set of films, indicating that GDH was equally distributed across the glass slides. Furthermore, no significant differences in absorbance (i.e., GDH activity) was found between the tested films (one-way ANOVA at α=0.01).


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) Box-and-whisker plot (Tukey test) of formazan absorbance as a measure of the activity of glutamate dehydrogenase (GDH) films on glass slides. GDH was evenly distributed over the glass slides (n=10 measurements for films 1 and 3, n=11 measurements for film 2). Dot in film 2 data respresents outlier. (B) Phosphate-activated glutaminase (PAG) activity in liver, brain and kidney sections in either the presence or absence of an auxiliary GDH film underneath the cryostat tissue sections. All absorbance measurements were corrected for nonspecific background staining in the absence of substrate. Activity is presented as the mean reaction velocity ± SEM (n=3).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3-0022155414551177: (A) Box-and-whisker plot (Tukey test) of formazan absorbance as a measure of the activity of glutamate dehydrogenase (GDH) films on glass slides. GDH was evenly distributed over the glass slides (n=10 measurements for films 1 and 3, n=11 measurements for film 2). Dot in film 2 data respresents outlier. (B) Phosphate-activated glutaminase (PAG) activity in liver, brain and kidney sections in either the presence or absence of an auxiliary GDH film underneath the cryostat tissue sections. All absorbance measurements were corrected for nonspecific background staining in the absence of substrate. Activity is presented as the mean reaction velocity ± SEM (n=3).
Mentions: To ascertain whether the auxiliary GDH activity of the GDH films between cryostat sections and glass slides were available throughout the tissue sections in a zero order manner (i.e., not rate limiting), we determined the distribution of GDH activity over the glass slides (Fig. 3A). GDH activity on the glass slides showed little variation over the entire set of films, indicating that GDH was equally distributed across the glass slides. Furthermore, no significant differences in absorbance (i.e., GDH activity) was found between the tested films (one-way ANOVA at α=0.01).

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