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

Abstract

Phosphate-activated glutaminase (PAG) converts glutamine to glutamate as part of the glutaminolysis pathway in mitochondria. Two genes, GLS1 and GLS2, which encode for kidney-type PAG and liver-type PAG, respectively, differ in their tissue-specific activities and kinetics. Tissue-specific PAG activity and its kinetics were determined by metabolic mapping using a tetrazolium salt and glutamate dehydrogenase as an auxiliary enzyme in the presence of various glutamine concentrations. In kidney and brain, PAG showed Michaelis-Menten kinetics with a K(m) of 0.6 mM glutamine and a V(max) of 1.1 µmol/mL/min when using 5 mM glutamine. PAG activity was high in the kidney cortex and inner medulla but low in the outer medulla, papillary tip, glomeruli and the lis of Henle. In brain tissue sections, PAG was active in the grey matter and inactive in myelin-rich regions. Liver PAG showed allosteric regulation with a K(m) of 11.6 mM glutamine and a V(max) of 0.5 µmol/mL/min when using 20 mM glutamine. The specificity of the method was shown after incubation of brain tissue sections with the PAG inhibitor 6-diazo-5-oxo-L-norleucine. PAG activity was decreased to 22% in the presence of 2 mM 6-diazo-5-oxo-L-norleucine. At low glutamine concentrations, PAG activity was higher in periportal regions, indicating a lower K(m) for periportal PAG. 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.

Keywords: enzyme histochemistry; enzyme inhibition; glutamate; glutamine; glutaminolysis; quantitation.

Figure 1.

Cellular carbohydrate metabolism. Abbreviations: ACLY, ATP citrate lyase; α-KG, α-ketoglutarate; α-KGDH, α-ketoglutarate dehydrogenase; CS, citrate synthase; FH, fumarate hydratase; GDH, glutamate dehydrogenase; IDH, isocitrate dehydrogenase; LDH, lactate dehydrogenase; MDH, malate dehydrogenase; ME, malic enzyme; PAG, phosphate-activated glutaminase; PDC, pyruvate dehydrogenase complex; SCS, succinyl coenzyme A synthetase; SDH, succinate dehydrogenase. (Reproduced from Botman et al. In Press in this issue).

Figure 2.

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.

Figure 3.

(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).

Figure 4.

(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).

Figure 5.

Phosphate-activated glutaminase (PAG) activity in brain tissue sections of a mouse in the presence of 0 mM, 0.5 mM, and 2 mM 6-diazo-5-oxo-L-norleucine (DON; PAG-specific inhibitor). PAG activity was inhibited dose-dependently by DON. All test reactions were corrected for nonspecific background staining in the absence of substrate. Error bars indicate SEM (n=10).

Figure 6.

Phosphate-activated glutaminase (PAG) activity in brain tissue sections. All test reactions were corrected for nonspecific background staining in the absence of substrate. For each glutamine concentration, three tissue sections per mouse were analyzed.

Figure 7.

(A) Kinetics of phosphate-activated glutaminase (PAG) activity in mouse kidney and brain tissue sections plotted against glutamine concentration. (B) Kinetics of PAG activity in mouse liver tissue sections plotted against various glutamine concentrations. Bars indicate SEM (n=3).

Figure 8.

Phosphate-activated glutaminase (PAG) activity staining in kidney tissue. (A) Stitched overview image of PAG activity in mouse kidney cryostat sections in the presence of 30 mM glutamine. Scale, 1 mm. (B) Example of PAG activity in a glomerulus. Scale, 50 µm. (C) PAG activity staining in the presence of 50 mM glutamine without glutamate dehydrogenase (GDH) underneath the tissue section. PAG activity was stained with GDH underneath the tissue sections in the presence of (D) 0 mM, (E) 2 mM, (F) 5 mM, (G) 10 mM, (H) 30 mM and (I) 50 mM glutamine. Boxes show measured (dotted line) and enlarged (continues line) areas in the specific tissue sections. Scale (C–I), 200 µm. Image stitching was performed using Adobe Photoshop CS6 (Adobe, San Jose, CA).

Figure 9.

Phosphate-activated glutaminase (PAG) activity staining in mouse brain cryostat sections. (A) Overview of PAG activity in mouse brain tissue in the presence of 5 mM glutamine. Scale, 0.5 mm. PAG activity was stained with glutamate dehydrogenase (GDH) underneath the tissue sections in the presence of (B) 0 mM, (C) 2 mM, (D) 5 mM, (E) 10 mM, (F) 30 mM and (G) 50 mM glutamine and (H) without GDH underneath the tissue section. Box shows the enlarged area in the tissue sections. Scale (B–H), 200 µm.

Figure 10.

Phosphate-activated glutaminase (PAG) activity staining in mouse liver cryostat sections. (A) Overview of PAG activity in mouse liver tissue in the presence of 30 mM glutamine. Scale, 0.5 mm. PAG activity was stained with glutamate dehydrogenase (GDH) underneath the tissue sections in the presence of (B) 0 mM, (C) 2 mM, (D) 5 mM, (E) 10 mM, (F) 30 mM and (G) 50 mM glutamine and (H) without GDH underneath the tissue section. Boxes show the measured (dotted line) and enlarged (continues line) areas in the tissue sections. Scale (B–H), 200 µm.

Figure 11.

Phosphate-activated glutaminase (PAG) activity in the presence of 37.5 mM or 30 mM glutamine (for liver, kidney and brain tissue) in various mouse tissues. All test reactions were corrected for nonspecific staining in the absence of substrate. Activity is presented as mean reaction velocity ± SEM (n=3).