Ammonia regulation of phosphate-activated glutaminase displays regional variation and impairment in the brain of aged rats

The regulation of PAG by ammonia in whole brain (Sprague-Dawley) and regional (Fischer-344) synaptosomal preparations from adult and aged animals was assessed. Whole brain synaptosomal preparations from both age groups displayed a significant decrease in PAG activity with increasing ammonium chloride concentrations, however, the aged rats exhibited a significant attenuation in ammonia-induced PAG inhibition. PAG activity measured in synaptosomes prepared from the striatum (STR), temporal cortex (TCX) and hippocampus (HIPP) was also inhibited by ammonium chloride. The STR showed the greatest degree of ammonia-induced PAG inhibition (55%) followed by the HIPP (30–35%) and the TCX (25–30%). This reduction in PAG activity was significantly attenuated in STR from aged rats at ammonium chloride concentrations greater than 50 M and in the TCX, PAG activity was significantly attenuated in the aged rats at ammonia concentrations of 0.5 and 1.0 mM. Ammonia regulation of PAG activity in the HIPP appeared to be unaffected by age. Ammonium chloride concentrations up to 5 mM had no effect on GLU release from cortical slices, although GLN efflux was significantly enhanced. These findings suggest that isozymes of PAG may exist in different brain regions based on their differential sensitivity to ammonia. The attenuation of ammonia-induced PAG inhibition seen in aged rats may have deleterious effects in the aged brain.Abbreviations PAG phosphate-activated glutaminase: L-glutamine amidohydrolase; EC 3.5.1.2 - STR striatum - TCX temporal cortex - HIPP hippocampus     

Regulation of phosphate-activated glutaminase (PAG) by glutamate analogues

The ability of structural analogues of glutamate (GLU) to modulate phosphate activated glutaminase (PAG) was assessed in the present series of studies. A number of GLU receptor agonists and antagonists were tested for their ability to inhibit synaptosomal PAG activity. PAG activity was determined by measuring GLU formation from 0.5mM glutamine (GLN) in the presence of 10 mM phosphate. GLU analogues at 5–10 mM were found to significantly inhibit PAG activity. It was determined that PAG inhibition occurred regardless of whether the GLU analogues were receptor agonists or antagonists, however, PAG inhibition was influenced by analogue chain length, isomeric form and substituent substitution. The glutamate uptake blockers, dihydrokainic acid and DL-threo--hydroxyaspartic acid were relatively weak inhibitors of PAG (<25% inhibition) as were the receptor agonists, ibotenic acid and (±)cis-2,3-piperidine-dicarboxylic acid. Other GLU analogues produced inhibition of PAG in the range of 40–70%. PAG inhibition by GLU analogues did not appear to differ substantially among the brain regions evaluated (cortex, striatum and hippocampus). The endogenous amino acids, glycine, taurine and N-acetylaspartic acid, also significantly inhibited PAG activity in the 5–10 mM range. The noncompetitive NMDA antagonists, (+)MK801 and ketamine, at a concentration of 5 mM, significantly stimulated PAG activity 1.5–2 fold over control values. The activation of PAG by (+)MK801 was dose-related, stereoselective and appeared to result from a synergistic interaction with phosphate to enhance substrate (GLN) binding to PAG. The results of these studies suggest that GLU analogues could potentially alter neurotransmitter GLU synthesis if sufficient concentrations of these drugs are used in in vitro or in vivo studies. Furthermore, preliminary evidence suggests that other endogenous amino acids (glycine, taurine, N-acetylaspartic acid) may modulate PAG activity. These studies have further characterized the structural requirements for the allosteric regulation of PAG by glutamate and its analogues.     

Kinetic properties of glutaminase from cerebral cortex

The rates of phosphate activated glutaminase activity in finely homogenised cerebral cortex and synaptosomes were measured. Activity was 25–50% higher at pH 7.0 than at pH 8.0. Glutamate inhibited activity with a K_i of 2–3 mM while aspartate had little effect. Calcium (1 mM) activated the enzyme but magnesium was without action. The pH profiles of the effects of these modulators of glutaminase activity in these finely ground preparations showed that all agents were more effective at pH 7.0 than at pH 8.0.Dedicated to Henry McIlwain.     

In vitro inhibition of brain phosphate-activated glutaminase by ammonia and manganese

IntroductionAs a consequence of the loss of liver function in chronic liver disease, increased levels of ammonia, manganese, and glutamine have been observed in the brain of hepatic encephalopathy patients.ObjectiveIn the present study, we explored phosphate activated glutaminase (PAG) activity in mitochondrial enriched fractions under treatment with ammonia and manganese.MethodsWe dissected out the brain cortex, striatum, and cerebellum of male Wistar rats 250−280 g weight; brain sections were pooled to obtain enriched mitochondrial fractions by differential centrifugation. Aliquots equivalent to 200 μg of protein were incubated with semi-log increasing concentrations of ammonia and/or manganese both as chloride salts (from 0 to 10 000 μM) and glutamine (4 mM) for 30 min. Then, the glutamate produced by the reaction was determined by HPLC coupled with fluorescence detection.Results and discussionBoth manganese and ammonia inhibited PAG in a concentration-dependent manner. Non-linear modeling was used to determine IC50 and IC20 for ammonia (120 μM) and manganese (2 mM). We found that PAG activity under the combination of IC20 of ammonia and manganese was equivalent to the sum of the effects of both substances, being PAG inhibition more pronounced in mitochondrial fractions from cerebellum. The PAG inhibition observed here could potentially explain a pathway for glutamine accumulation, by means of the inhibition of PAG activity as a consequence of increased concentrations of manganese and ammonia in the brain under liver damage conditions.     

Leucine: Effector of phosphate activated glutaminase in rat cerebral cortex

Phosphate activated glutaminase (PAG) was assayed in whole homogenate and synaptosomes of cerebral cortex from normal or fasted for 120 h rats. The specific activity (s.a.) of PAG was found diminished by 25% in the whole homogenate from the fasted animals compared to the normal. On the contrary, fasting did not affect PAG s.a. of the synaptosomal fraction. Reconstitution experiments showed that when the deproteinized supernatant of the 12,500g centrifugation from the fasted rats was added to the synaptosomes from either fed or fasted animals the PAG activity was diminished but there was no change when the corresponding supernatant from the fed animals was added to the synaptosomes from both conditions. When leucine at 5mM was added to the homogenate or to synaptosomes from fed or fasted animals the s.a. of PAG was significantly decreased. Even in the presence of aminooxyacetate the effect of leucine was observed. Branched chain amino acids i.e. leucine, isoleucine and valine at 0.5 mM each added to synaptosomes again decreased PAG activity. The addition of ketone bodies had no effect. It is suggested that leucine, because PAG has been implicated in the supply of transmitter glutamate, might be an important regulator of the pool of this neurotransmitter.     

Functional intracellular glutaminase activity in intact astrocytes

Numerous cellular metabolites such as glutamine, glutamate, phosphate, calcium, ammonia and acetyl derivatives are known to affect the phosphate-activated glutaminase activity in whole cell homogenates or extracts. Since measurements in extracts under non-physiological conditions may obscure the actual intracellular metabolic flux, the functional intracellular phosphate-activated glutaminase activity was measured by the formation of³H₂O froml-[2-³H]glutamine (Anal. Biochem. 127:134–142, 1982) in cultures of intact astrocytes, untreated and treated with dibutyryl c-AMP (DiBcAMP), in the presence of several potential effectors. These values were compared with enzyme levels determined in extracts from identical cells. The rate of¹⁴CO₂ release froml-[1-¹⁴C]glutamine was also measured in both untreated and DiBcAMP treated astrocytes. The intracellular activity of glutaminase for untreated cells assayed in MEM medium with 1mM radioactive glutamine was 88 nmol/mg protein/h and in DiBcAMP treated cells the rate was 153 nmol/mg protein/h. However, the enzymatic activity measured under optimal conditions in extracts from both untreated and treated cells was much higher, but essentially the same, about 1,750 nmol/mg protein/h. The rate of¹⁴CO₂ release froml-[1-¹⁴C]glutamine was 74 and 133 nmol/mg protein/h in untreated and DiBcAMP treated cells, respectively. This represents approximately 85% of the intracellular glutaminase activity. Furthermore, increasing the concentration of glutamine in the medium from 1 to 6.4 mM increased glutaminase intracellular activity about 3 fold in both untreated and treated cells. Addition of 250 M glutamate to the medium inhibited intracellular glutaminase activity by 70% under both treatment conditions. Deletion of glucose stimulated glutaminase activity. In contrast the removal of fetal bovine serum decreased activity by 35%. The addition of 10 mM phosphate and the alpha keto acids of isoleucine and valine marginally increased intracellular glutaminase activity. The addition of 0.4 mM ammonium chloride to the medium had no effect. An increase in media pH from 6.8 to 7.7 increased intracellular glutaminase activity almost 2 fold. These results provide evidence that phosphate-activated glutaminase activity in vivo is regulated by cellular metabolites, that its functional activity is 5–9% of the rate obtained using extracts, and this functional activity is sufficient to account for the rate of glutamine oxidation.Special issue dedicated to Dr. Elling Kvamme     

Effect of starvation or streptozotocin-diabetes on phosphate-activated glutaminase of different rat brain regions

Phosphate-activated glutaminase (PAG) was assayed in homogenates of brain cerebellum, hippocampus or striatum from normal, starved for 48 h or 120 h or streptozotocin-diabetic rats. Only the hippocampal enzyme was increased (47%) by diabetes. Starvation had no effect in any of the regions studied. PAG of synaptosomes or of non-synaptosomal mitochondria from the hippocampus was also increased by 48% and 22% respectively in diabetes. PAG of synaptosomes from the cortex, the cerebellum, or the striatum or of the non-synaptosomal mitochondria from the cortex were not affected by diabetes or prolonged (120h) starvation. A suggestion is presented that peripheral insulin, indirectly, may regulate PAG activity in a specific region of the rat brain.Abbreviations used PAG phosphate-activated glutaminase - LDH lactate dehydrogenase - s.a. specific activity     

Inhibition by glutamate of phosphate-dependent glutaminase of rat kidney.

A membrane-associated form of phosphate-dependent glutaminase was derived from sonicated mitochondria and purified essentially free of gamma-glutamyl transpeptidase activity. Increasing concentrations of phosphate cause a sigmoidal activation of the membrane-bound glutaminase. Phosphate also causes a similar effect on the rate of glutaminase inactivation by the two affinity labels, L-2-amino-4-oxo-5-chloropentanoic acid and 6-diazo-5-oxo-L-norleucine, as observed previously for the solubilized and purified enzyme. Therefore the two forms of glutaminase undergo similar phosphate-induced changes in conformation. A sensitive radioactive assay was developed and used to determine the kinetics of glutamate inhibition of the membrane-associated glutaminase. The Km for glutamine decreases from 36 to 4 mM when the phosphate concentration is increased from 5 to 100 mM. Glutamate is a competitive inhibitor with respect to glutamine at both high and low concentrations of phosphate. However, the Ki for glutamate is increased from 5 to 52 mM with increasing phosphate concentration. Therefore glutamine and glutamate interact with the same site on the glutaminase, but the specificity of the site is determined by the available phosphate concentration.     

Properties of phosphate activated glutaminase in astrocytes cultured from mouse brain

Astrocytes in primary cultures contain a relatively high activity, of phosphate activated glutaminase, although it is significantly lower than that of synaptosomal enriched preparations. The relatively high glutaminase activity in the astrocytes appears not to be caused by substrate induction, since a 10-fold variation in the glutamine concentration of the culture medium does not affect the activity. Of the reaction products, only glutamate inhibits astrocytic glutaminase whereas that of synaptosomal enriched preparations is inhibited by both glutamate and ammonia. Similar to the synaptosomal enzyme, glutaminase in astrocytes is inhibited about 50% by N-ethylmaleimide, indicating N-ethylmaleimide-sensitive and-insensitive compartments of the enzyme. Calcium activates glutaminase in astrocytes as in synaptosomes, by promoting phosphate activation. Except for the lower activity and the lack of effect of ammonia, the properties of the astroglial glutaminase has been found to be no different from that of the synaptosomal one. The relatively unrestrained astroglial glutaminase may, however, argue against the concept of a glutamine cycle operating in a stoichiometric manner.Abbreviations NEM N-ethylmaleimide - PAG Phosphate-activated glutaminase - PMB p-mercuribenzoate     

[3H]d-Aspartic acid release in brain slices of adult and aged fischer 344 rats

Alterations in glutamate content and uptake have been reported to occur in aged animals. The present studies used [³H]d-Aspartic acid ([³H]-D-ASP) release as a marker for glutamate neurotransmission. Frequency dependent [³H]-D-ASP release was measured in adult (8 month) and aged (28–30 month) Fischer 344 rats. Relatively high stimulation frequencies (>10 Hz) were required to induce [³H]-D-ASP release in both adult and aged F344 rats in temporal cortex and hippocampus. In both brain areas aged animals showed significantly more [³H]-D-ASP release than adult animals Kainic acid 1 mM failed to induce the release of [³H]-D-ASP in either temporal cortex or hippocampus. Omega conotoxin GVIA (5×10^–9M) a N and L type voltage sensitive calcium channel antagonist failed to inhibit [³H]-D-ASP stimulated release. These results demonstrate an increase in [³H]-D-ASP release in aged compared to adult F344 rats. The data also suggest a novel calcium channel may be involved in [³H]-D-ASP release.     

Evidence indicating that pig renal phosphate-activated glutaminase has a functionally predominant external localization in the inner mitochondrial membrane

Phosphate-activated glutaminase in intact pig renal mitochondria was inhibited 50-70% by the sulfhydryl reagents mersalyl and N-ethylmaleimide (0.3-1.0 mM), when assayed at pH 7.4 in the presence of no or low phosphate (10 mM) and glutamine (2 mM). However, sulfhydryl reagents added to intact mitochondria did not inhibit the SH-enzyme beta-hydroxybutyrate dehydrogenase (a marker of the inner face of the inner mitochondrial membrane), but did so upon addition to sonicated mitochondria. This indicates that the sulfhydryl reagents are impermeable to the inner membrane and that regulatory sulfhydryl groups for glutaminase have an external localization here. The inhibition observed when sulfhydryl reagents were added to intact mitochondria could not be attributed to an effect on a phosphate carrier, but evidence was obtained that pig renal mitochondria have also a glutamine transporter, which is inhibited only by mersalyl and not by N-ethylmaleimide. Mersalyl and N-ethylmaleimide showed nondistinguishable effects on the kinetics of glutamine hydrolysis, affecting only the apparent Vmax for glutamine and not the apparent Km calculated from linear Hanes-Woolf plots. Furthermore, both calcium (which activates glutamine hydrolysis), as well as alanine (which has no effect on the hydrolytic rate), inhibited glutamine transport into the mitochondria, indicating that transport of glutamine is not rate-limiting for the glutaminase reaction. Desenzitation to inhibition by mersalyl and N-ethylmaleimide occurred when the assay was performed under optimal conditions for phosphate activated glutaminase (i.e. in the presence of 150 mM phosphate, 20 mM glutamine and at pH 8.6). Desenzitation also occurred when the enzyme was incubated with low concentrations of Triton X-100 which did not affect the rate of glutamine hydrolysis. Following incubation with [14C]glutamine and correction for glutamate in contaminating subcellular particles, the specific activity of [14C]glutamate in the mitochondria was much lower than that of the surrounding incubation medium. This indicates that glutamine-derived glutamate is released from the mitochondria without being mixed with the endogenous pool of glutamate. The results suggest that phosphate-activated glutaminase has a functionally predominant external localization in the inner mitochondrial membrane.     

Response of rat liver glutaminase to pH. Mediation by phosphate and ammonium ions

The activity of rat liver glutaminase from sedimented fractions of freeze-thawed mitochondria is strongly affected by variation in pH over a physiologically relevant range at approximate physiological concentrations of activators. As pH increases from 7.1 to 7.7 at 0.7 mM ammonium and 10 mM phosphate, the S0.5 for glutamine decreases 3.5-fold, from 38 to 11 mM. This results in an 8-fold increase in reaction velocity at 10 mM glutamine. In addition, the M0.5 for phosphate activation decreases from 21 to 8.9 mM as pH increases from 7.1 to 7.7. This apparent effect of pH on the affinity of glutaminase for phosphate is similar to previous reports of the pH effect on activation by ammonium (Verhoeven, A. J., Van Iwaarden, J. F., Joseph, S. K., and Meijer, A. J. (1983) Eur. J. Biochem. 133, 241-244; McGivan, J. D., and Bradford, N. M. (1983) Biochim. Biophys. Acta 159, 296-302). Glutaminase does not respond to variation in pH between 7.1 and 7.7 when phosphate and ammonium are saturating. The effects of the two modifiers are additive. Each is still effective, as is pH, when the other is saturating. Therefore, it appears that the effects of pH on the apparent affinity of the enzyme for ammonium and phosphate account for the enzyme's response to pH. These results may help explain previous reports of minimal effects of pH on glutaminase at saturating concentrations of related substances (McGivan, J. D., Lacey, J. H., and Joseph, K. (1980) Biochim. J. 192, 537-542; Horowitz, M. L., and Knox, W. E. (1968) Enzymol. Biol. Clin. 9, 241-255; McGivan, J. D., and Bradford, N. M. (1983) Biochim. Biophys. Acta 759, 296-302). Glutaminase binds glutamine cooperatively with Hill coefficients ranging from 1.7 to 2.2, which suggests at least two and probably three or more interacting binding sites for glutamine. The strong response of liver glutaminase to pH and the fact that the reaction can supply metabolites for urea synthesis suggest a possible regulatory role of glutaminase in ureagenesis.     

Intracellular localization and properties of phosphate-dependent glutaminase in rat mesenteric lymph nodes.

Phosphate-dependent glutaminase was present at approximately similar activities in lymph nodes from mammals other than rat, and in thymus, spleen, Peyer's patches and bone marrow of the rat. This suggests that glutamine is important in all lymphoid tissues. Phosphate-dependent glutaminase activity was shown to be present primarily in the mitochondria of rat mesenteric lymph nodes, and most of the activity could be released by detergents. The properties of the enzyme in mitochondrial extracts were investigated. The pH optimum was 8.6 and the Km for glutamine was 2.0 mM. The enzyme was activated by phosphate, other phosphorylated compounds including phosphoenolpyruvate, and also leucine: 50% activation occurred at 5, 0.2 and 0.6 mM for phosphate, phosphoenolpyruvate and leucine respectively. The enzyme was inhibited by glutamate, 2-oxoglutarate, citrate and ammonia, and by N-ethylmaleimide and diazo-5-oxo-L-norleucine; 50% inhibition was observed at 0.7 and 0.1 mM for glutamate and 2-oxoglutarate respectively. Some of these properties may be important in the control of the enzyme activity in vivo.     

Regulatory effects of fatty acyl-coenzyme A derivatives on phosphate-activated pig brain and kidney glutaminase in vitro.

1. Fatty n-acyl-CoA derivatives in the concentration range 5muM-0.1mM and with 5-18 fatty acyl carbons have dual effects on phosphate-activated glutaminase from pig brain and kidney. Generally, fatty acyl-CoA derivatives in low concentrations activate the enzyme, but inhibit at higher concentrations; phosphate and citrate potentiate the activation, displaying positive co-operatively, and protect against inactivation. The fatty acyl-CoA derivatives affect glutaminase similarly to Bromothymol Blue, but differently from acetyl-CoA, which activates the enzyme only at very low phosphate or citrate concentrations. 2. Saturated fatty acyl-CoA derivatives, with 5-10 fatty acyl carbons, only activate the enzyme in the concentration range 0-0.1 mM. When the fatty acyl chain is elongated, the fatty acyl-CoA derivatives gradually become more powerful inhibitors of glutaminase at the expense of their activating capacity. In particular, palmitoyl-CoA and stearoyl-CoA are strong inhibitors at concentrations (10 muM) at which the corresponding free fatty acids and fatty acyl-carnitine derivatives have no effect. 3. The unsaturated fatty acyl-CoA derivatives, oleoyl-CoA and linoleoyl-CoA, behave as potent activators in the lower part of the concentration range tested (0-0.05mM), and as inhibitors in the upper part of this range (0.02-0.10mM). Oleic acid and linoleic acid have similar properties, but their activating capacity is less pronounced. 4. Phosphate both prevented and reversed the inhibition, but no restoration of activity was possible once the enzyme became inactivated. 5. By changing the pH from 7.0 to 8.0 the activating capacity of the fatty acyl-CoA derivatives is increased, as is their concentration range for activation. 6. The fatty acyl-CoA derivatives are somewhat more potent activator for brain glutaminase, but otherwise they affect the two enzymes similarly.     

Variations in the kinetic response of several different phosphate-dependent glutaminase isozymes during acute metabolic acidosis

Summary We describe the kinetic modifications to mitochondrial-membrane-bound phosphate-dependent glutaminase in various types of rat tissue brought about by acute metabolic acidosis. The activity response of phosphate-dependent glutaminase to glutamine was sigmoidal, showing positive co-operativity, the Hill coefficients always being higher than 2. The enzyme from acidotic rats showed increased activity at subsaturating concentrations of glutamine in kidney tubules, as might be expected, but not in brain, intestine or liver tissues. Nevertheless, when brain and intestine from control rats were incubated in plasma from acutely acidotic rats enzyme activity increased at 1 mM glutamine in the same way as in kidney cortex. The enzyme from liver tissue remained unaltered. S_0.5 and n_H values decreased significantly in kidney tubules, enterocytes and brain slices preincubated in plasma from acidotic rats. The sigmoidal curves of phosphate-dependent glutaminase shifted to the left without any significant changes in V_max. The similar response of phosphate-dependent glutaminase to acute acidosis in the kidney, brain and intestine confirms the fact that enzymes from these tissues are kinetically identical and reaffirms the presence of an ammoniagenic factor in plasma, either produced or concentrated in the kidneys of rats with acute acidosis.Abbreviations Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid - EDTA NN-1,2-Ethane-diylbis [N-(carboxymethyl)glycyne] - Tris 2-amino-2-hydroxymethyl-1,3-propanediol - PDG phosphate dependent glutaminase Publication No. 145 from Drogas, Tóxicos Ambientales y Metabolismo Celular Research Group. Department of Biochemistry and Molecular Biology, University of Granada, Spain     

Functional characterization of a salt- and thermotolerant glutaminase from Lactobacillus rhamnosus

The deamination of glutamine is a crucial step in the production of enzymatically hydrolyzed plant proteins to reach high glutamic acid yields. The required glutaminase activity usually is provided by addition of technical enzymes or by in situ generation from fungi, yeast or bacteria (i.e. Aspergillus oryzae in soy sauce production). We screened food-grade Lactobacilli for potential glutaminase activity and selected the enzyme found in Lactobacillus rhamnosus for further characterization. Glutaminase from L. rhamnosus was induced by growing the microorganism on hydrolyzed wheat gluten, a glutamine-rich protein source. Glutamine deaminating activity (glutaminase, EC 3.5.1.2) was found to be membrane-bound and lost its activity gradually upon solubilization. Functional studies of the glutaminase showed an optimal working pH of 7.0 and maximum activity at 50 °C. High salt-tolerance of the enzyme was observed, i.e. the presence of 5% (w/v) salt increased glutaminase activity almost two-fold and 90% of the initial activity still remained at 15% (w/v) salt. The glutaminase activity showed typical Michaelis–Menten behavior with an affinity constant K_m of 4.8±0.4 mM for glutamine and a V_max of 101±2 U/l.     

The orientation of phosphate-dependent glutaminase on the inner membrane of rat renal mitochondria

Phosphate-dependent glutaminase is associated with the inner membrane of rat renal mitochondria. The orientation of this enzyme was characterized by comparing its sensitivity in isolated mitochondria and in mitoplasts to two membrane impermeable inhibitors. Mitoplasts were prepared by repeated swelling of mitochondria in a hypotonic phosphate solution. This procedure released greater than 70% of the adenylate kinase from the intermembrane space, but less than 10 and 25% of the marker activities characteristic of the inner membrane and matrix compartments, respectively. The addition of 20 microM p-chloromercuriphenylsulfonate (pCMPS) caused a rapid inactivation of the purified glutaminase. In contrast, the glutaminase contained in isolated mitochondria and mitoplasts was only slightly affected by the addition of up to 2 mM pCMPS. Similarly, the activity in mitochondria and mitoplasts was not inhibited by the addition of an excess of inactivating Fab antibodies. However, a similar extent of inactivation occurred when either membrane fraction was incubated with concentrations of octylglucoside greater than 0.35%. Mitochondria were also treated with increasing concentrations of digitonin. At 0.4 mg digitonin/mg protein, all of the adenylate kinase was released but the glutaminase activity was either slightly inhibited or unaffected by the addition of pCMPS or the Fab antibodies, respectively. These studies establish that the glutaminase is localized on the inner surface of the inner membrane. Therefore, mitochondrial catabolism of glutamine must occur only within the matrix compartment.     

Neuronal Glutamine Utilization: Glutamine/Glutamate Homeostasis in Synaptosomes

The synaptosomal metabolism of glutamine was studied under in vitro conditions that simulate depolarization in vivo. With [2-15N]glutamine as precursor, the [glutamine]i was diminished in the presence of veratridine or 50 mM KCl, but the total amounts of [15N]glutamate and [15N]aspartate formed were either equal to those of control incubations (veratridine) or higher (50 mM [KCl]). This suggests that depolarization decreases glutamine uptake and independently augments glutaminase activity. Omission of sodium from the medium was associated with low internal levels of glutamine which indicates that influx occurs as a charged Na(+)-amino acid complex. It is postulated that a reduction in membrane potential and a collapse of the Na+ gradient decrease the driving forces for glutamine accumulation and thus inhibit its uptake and enhance its release under depolarizing conditions. Inorganic phosphate stimulated glutaminase activity, particularly in the presence of calcium. At 2 mM or lower [phosphate] in the medium, calcium inhibited glutamine utilization and the production of glutamate, aspartate, and ammonia from glutamine. At a high (10 mM) medium [phosphate], calcium stimulated glutamine catabolism. It is suggested that a veratridine-induced increase in intrasynaptosomal inorganic phosphate is responsible for the enhancement of flux through glutaminase; calcium affects glutaminase indirectly by modulating the level of free intramitochondrial [phosphate]. Because phosphate also lowers the Km of glutaminase for glutamine, augmentation of the amino acid breakdown may occur even when depolarization lowers [glutamine]i. Reducing the intrasynaptosomal glutamate to 26 nmol/mg of protein had little effect on glutamine catabolism, but raising the pH to 7.9 markedly increased formation of glutamate and aspartate. It is concluded that phosphate and H+ are the major physiologic regulators of glutaminase activity.     

Activity of renal enzymes producing ammonia from glutamine in the absence of phosphate: 2. Effect of phosphate dependent glutaminase inhibition by heating

It is known that heating at 50 degrees C for 10 minutes inhibits phosphate dependent glutaminase (PDG) activity of renal cortex, without any effect on gamma-glutamyl transpeptidase (gamma GT) and its phosphate independent glutaminase (PIG) activity. The effect of heating on PIG and total gamma GT activities was evaluated in renal cortex homogenates of rats both in normal acid-base equilibrium and in chronic metabolic acidosis (CMA). Homogenates were incubated in a medium containing glutamine 2 mM, no phosphate, at pH 7,40. PIG activity was measured as glutamate production and total gamma GT activity as ammonia production. In normal rats PIG activity was unchanged after heating, whereas a significant decrease of total gamma GT activity was observed (p less than 0,01). CMA caused an increase in both PIG and total gamma GT activity (p less than 0,01) and these increased to a further extent after heating. In both normal and acidotic rats the glutamate production/ammonia production ratio rose to about 1. In conclusion: a) in the experimental setting used for this study PDG activity does not intervene in glutamate and ammonia production from glutamine; b) heating causes an inhibition of gamma GT activities, other than PIG, both in normal and in acidotic rats; c) in CMA heating increases PIG activity of gamma GT.     

Renal ammoniagenesis in rats made acutely acidotic by swimming

Rats develop metabolic acidosis acutely after exercise by swimming. Renal cortical slices from exercised rats show an increase in both ammoniagenesis and gluconeogenesis from glutamine. In addition, plasma from the exercised rats also stimulates ammoniagenesis in renal cortical slices from normal rats. In exercised rats renal phosphate dependent glutaminase shows a 200% activation when the enzyme activity is measured at subsaturating concentration of glutamine (1 mM) while only an increase of 12% in Vmax is observed. When kidney slices from normal rats are incubated in plasma from exercised rats an activation of phosphate dependent glutaminase is obtained with a 1.0 mM (100%) but not with 20 mM glutamine as substrate. This activation of phosphate dependent glutaminase at subsaturating levels of substrate may indicate a conformational change in PDG effected by a factor present in the plasma of exercised acidotic rats.