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.     

Novel Form of Phosphate Activated Glutaminase in Cultured Astrocytes and Human Neuroblastoma Cells,PAG in Brain Pathology and Localization in the Mitochondria

A novel form of phosphate activated glutaminase (PAG), catalyzing the synthesis of glutamate from glutamine, has been detected in cultured astrocytes and SH-SY5Y neuroblastoma cells. This enzyme form is different from that of the kidney and liver isozymes. In these cells we found high enzyme activity, but no or very weak immunoreactivity against the kidney type of PAG, and no immunoreactivity against the liver type. PAG was also investigated in brain under pathological conditions. In patients with Down's syndrome the immunoreactivity in the frontoparietal cortex was significantly reduced. The findings leading to our conclusion of a functionally active PAG on the outer face of the inner mitochondrial membrane are discussed, and a model is presented.     

Possible mechanisms of the efflux of glutamate from kidney mitochondria generated by the activity of mitochondrial glutaminase.

The transport of glutamate across the inner membrane of kidney mitochondria and the influx of glutamine into the mitochondria was studied using an oxygen electrode, the swelling technique and by continous recording of the activity of the mitochondrial glutaminase by an NH4+-sensitive electrode. It is well known that the enzyme is activated by inorganic phosphate and strongly inhibited by glutamate. 1. Avenaciolide, Bromocresal purple and Bromothymol blue inhibited the respiration of the mitochondria almost completely in the presence of glutamate as substrate but not in the presence of glutamine. Production of aspartate during the oxidation of glutamine was not significantly inhibited by avenaciolide but it was markedly suppressed by Bomocresol purple and Bromothymol blue. 2. Swelling of kidney mitochondria in an isosmotic solution of glutamine and ammonium phosphate was not inhibted by avenaciolide or Bromocresol purple indicating that these substances do not inhibit the penetration of the mitochondrial membrane by glutamine or phosphate. 3. The activity of the mitochondrial glutaminase was strongly inhibited by avenaciolide or Bromocresol purple in the presence of inhibitos of respiration or an uncoupler but not in ther absence. Experimental data suggest that this was caused by the inhibition of glutamate efflux. The addition of a detergent removed this inhibition. On the basis of these observations it was concluded that two mechanisms exist which enable glutamate to leave the inner space of kidney mitochondria: (a) an electrogenic efflux coupled to the respiration-driven proton translocation and the presence of a membrane potential (positive outside) and (b) an electroneutral glutamate-hydroxyl antiporter which is inhibted by avenaciolide and which operates in both directions. Our observations do not support the existence of the electrogenic glutamine-glutamate antiporter or glutamate-aspartate exchange in the mitochondria studied.     

Purification of phosphate-dependent glutaminase from isolated mitochondria of Ehrlich ascites-tumour cells.

Phosphate-dependent glutaminase was purified to homogeneity from isolated mitochondria of Ehrlich ascites-tumour cells. The enzyme had an Mr of 135,000 as judged by chromatography on Sephacryl S-300. SDS/polyacrylamide-gel electrophoresis displayed two protein bands, with Mr values of 64,000 and 56,000. Two major immunoreactive peptides of Mr values of 65,000 and 57,000 were found by immunoblot analysis using anti-(rat kidney glutaminase) antibodies. The concentration-dependences for both glutamine and phosphate were sigmoidal, with S0.5 values of 7.6 mM and 48 mM, and Hill coefficients of 1.5 and 1.6, respectively. The glutaminase pH optimum was 9. The activation energy of the enzymic reaction was 58 kJ/mol. The enzyme showed a high specificity towards glutamine. A possible explanation for the different kinetic behaviour found for purified enzyme and for isolated mitochondria [Kovacević (1974) Cancer Res. 34, 3403-3407] should be that a conformational change occurs when the enzyme is extracted from the mitochondrial inner membrane.     

Importance of the flux of phosphate across the inner membrane of kidney mitochondria for the activation of glutaminase and the transport of glutamine

The effect of mersalyl, an inhibitor of phosphate transport across the inner mitochondrial membrane, was investigated on the uncoupled respiration of pig kidney mitochondria in the presence of glutamine as substrate and on the activity of the phosphate-dependent glutaminase in the intact organelles. In addition, the submitochondrial location of the enzyme was reinvestigated.

1. (1) It was found that mersalyl completely inhibits uncoupled respiration of the mitochondria in the presence of glutamine as substrate, whereas respiration with glutamate was not affected. The same amount of mersalyl which inhibits coupled oxidation of glutamine also inhibits coupled oxidation of glutamate and some other substrates.

2. (2) Mersalyl strongly inhibited the activation of glutaminase in intact mitochondria only in the presence of inhibitors of electron transport or of an uncoupler. The addition of a detergent prevented or fully released the inhibition. The effect of mersalyl was observed even when the mitochondria were pre-incubated with phosphate or incubated in the phosphate-free medium. If mersalyl and carbonyl cyanide m-chlorophenylhydrazone (CCCP) were added 3 min after pre-incubation with phosphate the same intramitochondrial concentration of the anion as in control experiments was found, whereas the activity of glutaminase was severely inhibited. These findings suggest that the activation of the enzyme by phosphate in intact nonenergized mitochondria occurs only if the activator moves across the inner mitochondrial membrane.

3. (3) Mersalyl (plus CCCP) markedly decreased [¹⁴C]glutamine- and [³²P]-phosphate-permeable mitochondrial spaces. A close correlation between the decrease of phosphate and glutamine permeable spaces and the inhibition of glutaminase activity was found.

4. (4) If the activation energy of the enzyme was determined with frozen mitochondrial preparations, a discontinuity or break in the Arrhenius plot was observed, whereas the presence of a detergent completely abolished the break. Digitonin or ultrasonic treatment of the mitochondria followed by separation of the membrane and the soluble fraction revealed that glutaminase is a membrane-bound enzyme.

On the basis of these findings it is concluded that there is an association between the transport of phosphate on one side and the transport of glutamine and glutaminase activity on the other. It is possible that the movement of phosphate across the membrane activates the enzyme which facilitates diffusion of glutamine down a concentration gradient. However, the existence of a specific glutamine-phosphate carrier is not ruled out.     

Phosphate-Activated Glutaminase and Mitochondrial Glutamine Transport in the Brain

A review of the properties of purified and tissue bound phosphate activated glutaminase (PAG) in brain and kidney (pig and rat) is presented, based on kinetic, electron microscopic and immunocytochemical studies. PAG is a mitochondrial enzyme and two pools can be separated, a soluble and membrane associated one. Intact mitochondria appear to express PAG accessible only to the outer phase of the inner mitochondrial membrane. This PAG has properties similar to that of the membrane fraction and polymeric form of purified enzyme. PAG in the soluble fraction has properties similar to that of the monomeric form of purified enzyme and is assumed to be dormant due to the high matrix concentration of the inhibitor glutamate. A hypothetical model for the localization of PAG in the mitochondria is presented. The activity of PAG in vivo is assumed to be regulated by cytosolic glutamate and other compounds, that affect the activation by phosphate. Glutamine is transported into brain and kidney mitochondria by a protein catalyzed energy requiring process, which may be mediated by more than one protein. There is no correlation between glutamine hydrolysis and transport.     

Conditions for activity of glutaminase in kidney mitochondria

1. Rat kidney mitochondria oxidize glutamate very slowly. Addition of glutamine stimulates this respiration two- to three-fold. Addition of glutamate also stimulates respiration in the presence of glutamine. 2. By measuring mitochondrial swelling in iso-osmotic solutions of glutamine or of ammonium glutamate it was shown that glutamine penetrates the mitochondrial membrane rapidly whereas ammonium glutamate penetrates very slowly. 3. Experiments in which reduction of NAD(P)⁺ was measured in preparations of intact and broken mitochondria indicated that glutamate dehydrogenase shows the phenomenon of `latency'. On the addition of glutamine rapid reduction of nicotinamide nucleotides in intact mitochondria was obtained. 4. During the action of glutaminase there is an accumulation of glutamate inside the mitochondria. 5. When the mitochondria were suspended in a medium containing glutamine, P_i and rotenone the rate of production of ammonia was stimulated by the addition of a substrate, e.g. succinate. Addition of an uncoupler or antimycin A abolished this stimulation. 6. The effects of succinate and uncoupler were especially pronounced in the presence of glutamate, which is an inhibitor of glutaminase activity by competition with P_i. 7. Determination of the enzyme activity in media at different pH values showed that the optimum pH for glutaminase activity in the preparation of broken mitochondria was 8, whereas for intact mitochondria it was dependent on the energy state. In the presence of succinate as an energy source it was pH 8.5, but in the presence of uncoupler or antimycin A it was 9. This displacement of the pH optimum to a higher value was especially pronounced in the presence of both glutamate and uncoupler. 8. If nigericin was present in potassium chloride medium the pH optimum for enzyme activity in intact non-respiring mitochondria was nearly the same as in the preparation of broken mitochondria; however, its presence in K⁺-free medium displaced the pH optimum for glutaminase activity to a very high value. 9. It is postulated that because of low permeability of the kidney mitochondrial membrane to glutamate the latter accumulates inside the mitochondria, and that this leads to the inhibition of the enzyme by competition with P_i and also by lowering the pH of the intramitochondrial space. With succinate as substrate for respiration there is an outward translocation of H⁺ ions, which together with accumulation of P_i increases glutaminase activity. Translocation of K⁺ ions inward increases the enzyme activity, perhaps by increasing the pH of the internal spaces and causing an accumulation of P_i. 10. The importance of the location of the enzyme in the mitochondria in relation to its biological function and conditions for activity is discussed.     

Properties and submitochondrial localization of pig and rat renal phosphate-activated glutaminase

Two pools ofphosphate-activated glutaminase (PAG) were separated from pig and ratrenal mitochondria. The partition of enzyme activity corresponded withthat of the immunoreactivity and also with the postembedding immunogoldlabeling of PAG, which was associated partly with the inner membraneand partly with the matrix. The outer membrane was not labeled. PAG inintact mitochondria showed enzymatic characteristics that were similarto that of the membrane fraction and also mimicked that of thepolymerized form of purified pig renal PAG. PAG in the soluble fractionshowed properties similar to that of the monomeric form of purifiedenzyme. It is indicated that the pool of PAG localized inside the innermitochondrial membrane is dormant due to the presence of highconcentrations of the inhibitor glutamate. Thus the enzymaticallyactive PAG is assumed to be localized on the outer face of the innermitochondrial membrane. The activity of this pool of PAG appears to beregulated by compounds in the cytosol, of which glutamate may be most important.

     

Possible mechanisms of the efflux of glutamate from kidney mitochondria generated by the activity of mitochondrial glutaminase

The transport of glutamate across the inner membrane of kidney mitochondria and the influx of glutamine into the mitochondria was studied using an oxygen electrode, the swelling technique and by continous recording of the activity of the mitochondrial glutaminase by an NH₄⁺-sensitive electrode. It is well known that the enzyme is activated by inorganic phosphate and strongly inhibited by glutamate.

1. 1. Avenaciolide, Bromocresal purple and Bromothymol blue inhibited the respiration of the mitochondria almost completely in the presence of glutamate as substrate but not in the presence of glutamine. Production of aspartate during the oxidation of glutamine was not significantly inhibited by avenaciolide but it was markedly suppressed by Bomocresol purple and Bromothymol blue.

2. 2. Swelling of kidney mitochondria in an isosmotic solution of glutamine and ammonium phosphate was not inhibited by avenaciolide or Bromocresol purple indicating that these substances do not inhibit the penetration of the mitochondrial membrane by glutamine or phosphate.

3. 3. The activity of the mitochondrial glutaminase was strongly inhibited by avenaciolide or Bromocresol purple in the presence of inhibitors of respiration or an uncoupler but not in their absence. Experimental data suggest that this was caused by the inhibition of glutamate efflux. The addition of a detergent removed this inhibition.

On the basis of these observations it was concluded that two mechanisms exist which enable glutamate to leave the inner space of kidney mitochondria: (a) an electrogenic efflux coupled to the respiration-driven proton translocation and the presence of a membrane potential (positive outside) and (b) an electroneutral glutamate-hydroxyl antiporter which is inhibited by avenaciolide and which operates in both directions. Our observations do not support the existence of the electrogenic glutamine-glutamate antiporter or glutamate-aspartate exchange in the mitochondria studied.     

Glutamine synthetase and glutaminase activities in various hepatoma cells

Glutamine synthetase and glutaminase activities in a series of hepatoma cells of human and rat origins were determined for comparison with normal liver tissues. Marked decrease in glutamine synthetase activity was observed in the tumor cells. Phosphate-dependent and phosphate-independent glutaminase activities were increased compared with those from normal liver tissues. Well coupled mitochondria were isolated from HuH 13 line of human hepatoma cells and human liver. Oxypolarographic tests showed that glutamine oxidation was prominent in the tumor mitochondria, while mitochondria from the liver showed a feeble glutamine oxidation. Glutamine oxidation was inhibited by prior incubation of the mitochondria with DON (6-diazo-5-oxo-L-norleucine), which inhibited mitochondrial glutaminase. These results indicate that the product of glutamine hydrolysis, glutamate, is catabolized in the tumor mitochondria to supply ATP.     

Amino acid concentrations and selected enzyme activities in rat auditory,olfactory, and visual systems

Homogenates of specific brain regions of three sensory systems (auditory, olfactory, and visual) were prepared from pigmented Long-Evans Hooded rats and assayed for amino acid concentrations and activities of glutaminase, aspartate aminotransferase (total, cytosolic, and, by difference, mitochondrial), malate dehydrogenase, lactate dehydrogenase, and choline acetyltransferase. Comparing the quantitative distributions among regions revealed significant correlations between AAT and aspartate, between glutaminase and glutamate, between glutamate and glutamine, and between AAT plus glutaminase, or glutaminase alone, and the sum of aspartate, glutamate, and GABA, suggesting a metabolic pathway involving the synthesis of a glutamate pool as precursor to aspartate and GABA. Of the inhibitory transmitter amino acids, GABA concentrations routinely exceeded those of glycine, but glycine concentrations were relatively high in brainstem auditory structures.     

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.     

Transport of glutamine and glutamate in kidney mitochondria in relation to glutamine deamidation

1. In the absence of added ADP glutamine is transformed by pig kidney mitochondria to ammonium glutamate, which appears in the external medium. This reaction is stimulated only slightly by the addition of ADP, but under these conditions about 20% of the glutamate is oxidized to aspartate. 2. Externally added glutamate is oxidized to aspartate, and at about the same rate as glutamine. 3. The net rates of glutamine and glutamate influx into the intramitochondrial compartment are very slow. 4. The phosphate-dependent glutaminase activity of intact mitochondria is stimulated by the provision of energy. 5. The provision of energy also decreases the concentration of glutamate and increases the concentration of glutamine in the intramitochondrial compartment. These energy-linked changes in the glutamine and glutamate concentrations are of equal magnitude. 6. It is suggested that transport of glutamine and glutamate across the inner membrane of kidney mitochondria occurs by an obligatory exchange between the two metabolites, and is electrogenic. The existence of an electrogenic glutamine-glutamate anti-porter is proposed.     

The pathway of glutamine and glutamate oxidation in isolated mitochondria from mammalian cells

1. Pyruvate strongly inhibited aspartate production by mitochondria isolated from Ehrlich ascites-tumour cells, and rat kidney and liver respiring in the presence of glutamine or glutamate; the production of (14)CO(2) from l-[U-(14)C]glutamine was not inhibited though that from l-[U-(14)C]glutamate was inhibited by more than 50%. 2. Inhibition of aspartate production during glutamine oxidation by intact Ehrlich ascites-tumour cells in the presence of glucose was not accompanied by inhibition of CO(2) production. 3. The addition of amino-oxyacetate, which almost completely suppressed aspartate production, did not inhibit the respiration of the mitochondria in the presence of glutamine, though the respiration in the presence of glutamate was inhibited. 4. Glutamate stimulated the respiration of kidney mitochondria in the presence of glutamine, but the production of aspartate was the same as that in the presence of glutamate alone. 5. The results suggest that the oxidation of glutamate produced by the activity of mitochondrial glutaminase can proceed almost completely through the glutamate dehydrogenase pathway if the transamination pathway is inhibited. This indicates that the oxidation of glutamate is not limited by a high [NADPH]/[NADP(+)] ratio. 6. It is suggested that under physiological conditions the transamination pathway is a less favourable route for the oxidation of glutamate (produced by hydrolysis of glutamine) in Ehrlich ascites-tumour cells, and perhaps also kidney, than the glutamate dehydrogenase pathway, as the production of acetyl-CoA strongly inhibits the first mechanism. The predominance of the transamination pathway in the oxidation of glutamate by isolated mitochondria can be explained by a restricted permeability of the inner mitochondrial membrane to glutamate and by a more favourable location of glutamate-oxaloacetate transaminase compared with that of glutamate dehydrogenase.     

Identification of stage-specific markers during differentiation of hair cells from mouse inner ear stem cells or progenitor cells in vitro

The induction of inner ear hair cells from stem cells or progenitor cells in the inner ear proceeds through a committed inner ear sensory progenitor cell stage prior to hair cell differentiation. To increase the efficacy of inducing inner ear hair cell differentiation from the stem cells or progenitor cells, it is essential to identify comprehensive markers for the stem cells/progenitor cells from the inner ear, the committed inner ear sensory progenitor cells and the differentiating hair cells to optimize induction conditions. Here, we report that we efficiently isolated and expanded the stem cells or progenitor cells from postnatal mouse cochleae, and induced the generation of inner ear progenitor cells and subsequent differentiation of hair cells. We profiled the gene expression of the stem cells or progenitor cells, the inner ear progenitor cells, and hair cells using aRNA microarray analysis. The pathway and gene ontology (GO) analysis of differentially expressed genes was performed. Analysis of genes exclusively detected in one particular cellular population revealed 30, 38, and 31 genes specific for inner ear stem cells, inner ear progenitor cells, and hair cells, respectively. We further examined the expression of these genes in vivo and determined that Gdf10+Ccdc121, Tmprss9+Orm1, and Chrna9+Espnl are marker genes specific for inner ear stem cells, inner ear progenitor cells, and differentiating hair cells, respectively. The identification of these marker genes will likely help the effort to increase the efficacy of hair cell induction from the stem cells or progenitor cells.     

Glutamine from Glial Cells Is Essential for the Maintenance of the Nerve Terminal Pool of Glutamate: Immunogold Evidence from Hippocampal Slice Cultures

Abstract: The immunogold labeling for glutamate and glutamine was studied at the electron microscopic level in hippocampal slice cultures following inhibition of l -glutamine synthetase [ l -glutamate:ammonia ligase (ADP-forming); EC 6.3.1.2]. In control cultures, glutamate-like immunoreactivity was highest in terminals, intermediate in pyramidal cell bodies, and low in glial cells. Glutamine-like immunoreactivity was high in glial cells, intermediate in pyramidal cell bodies, and low in terminals. After inhibition of glutamine synthetase with l -methionine sulfoximine, glutamate-like immunoreactivity was reduced by 52% in terminals and increased nearly fourfold in glia. Glutamine-like immunoreactivity was reduced by 66% in glia following l -methionine sulfoximine, but changed little in other compartments. In cultures that were treated with both l -methionine sulfoximine and glutamine (1.0 m M ), glutamate-like immunoreactivity was maintained at control levels in terminals, whereas in glia glutamate-like immunoreactivity was increased and glutamine-like immunoreactivity was decreased to a similar extent as in cultures treated with l -methionine sulfoximine alone. We conclude that (a) glutamate accumulates in glia when the flux through glutamine synthetase is blocked, emphasizing the importance of this pathway for the handling of glutamate; and (b) glutamine is necessary for the maintenance of a normal level of glutamate in terminals, and neither reuptake nor de novo synthesis through pathways other than the glutaminase reaction is sufficient.     

Developmental change of endogenous glutamate and gamma-glutamyl transferase in cultured cerebral cortical interneurons and cerebellar granule cells,and in mouse cerebral cortex and cerebellum in vivo

The developmental change of endogenous glutamate, as correlated to that of gamma-glutamyl transferase and other glutamate metabolizing enzymes such as phosphate activated glutaminase, glutamate dehydrogenase and aspartate, GABA and ornithine aminotransferases, has been investigated in cultured cerebral cortex interneurons and cerebellar granule cells. These cells are considered to be GABAergic and glutamatergic, respectively. Similar studies have also been performed in cerebral cortex and cerebellum in vivo. The developmental profiles of endogenous glutamate in cultured cerebral cortex interneurons and cerebellar granule cells corresponded rather closely with that of gamma-glutamyl transferase and not with other glutamate metabolizing enzymes. In cerebral cortex and cerebellum in vivo the developmental profiles of endogenous glutamate, gamma-glutamyl transferase and phosphate activated glutaminase corresponded with each other during the first 14 days in cerebellum, but this correspondence was less good in cerebral cortex. During the time period from 14 to 28 days post partum the endogenous glutamate concentration showed no close correspondence with any particular enzyme. It is suggested that gamma-glutamyltransferase regulates the endogenous glutamate concentration in culture neurons. The enzyme may also be important for regulation of endogenous glutamate in brain in vivo and particularly in cerebellum during the first 14 days post partum. Gamma-glutamyl transferase in cultured neurons and brain tissue in vivo appears to be devoid of maleate activated glutaminase.Abbreviations used Asp-T aspartate aminotransferase (EC 2.6.1.1) - GABA-T GABA aminotransferase (EC 2.6.1.19) - GAD glutamate decarboxylase (EC 4.1.1.15) - gamma-GT gamma-glutamyl transferase (gamma-glutamyl transpeptidase) (EC. 2.3.2.2) - Glu glutamate - GDH glutamate dehydrogenase (EC 1.4.1.3) - GS glutamine synthetase (EC 6.3.1.2) - MAG maleate activated glutaminase - Orn-T ornithine aminotransferase (EC 2.6.1.13) - PAG phosphate activated glutaminase (EC 3.5.1.1)     

beta-2-Aminobicyclo-(2.2.1)-heptane-2-carboxylic acid. A new activator of glutaminase in intact rat liver mitochondria

beta-(+/-)-2-Aminobicyclo-(2.2.1)-heptane-2-carboxylic acid (BCH) stimulated, in a concentration-dependent manner, the formation of glutamate by mitochondria isolated from rat liver and incubated with 20 mM glutamine. Maximum enhancement was seen with 10 mM BCH while 5 mM leucine was without effect. The initial lag in the rate of glutamate formation was not eliminated by BCH. Preincubation of the mitochondria without glutamine also did not abolish the lag period; to the contrary, it resulted in a progressive deactivation of the glutaminase. The decrease in enzyme activity during the preincubation without glutamine was partially reversed by the addition of either 10 mM BCH or 1.4 mM NH4Cl and was essentially abolished by their combined action. The apparently sigmoid rise in the activity of glutaminase with increasing concentration of glutamine became hyperbolic in the presence of 1.4 mM NH4Cl. BCH stimulated the NH4Cl-activated glutaminase in the entire range of glutamine concentrations studied (2-40 mM) without changing the S50 value. In mitochondria disrupted by repeated cycles of freezing and thawing, the enzymatic activity was maximal even in the absence of BCH. It is postulated that BCH is a potent activator of mitochondrial glutaminase and that manifestation of its action requires intact organelle structure. In addition, it is concluded that BCH-induced stimulation of glutamine catabolism in isolated hepatocytes (Zaleski, J., Wilson, D. F., and Erecinska, M. (1986) J. Biol. Chem. 261, 14082-14090) is the consequence of activation of the mitochondrial glutaminase.