Phosphate activated glutaminase-like immunoreactivity in the nervous system from different species and in different neuronal cell types and in astrocytes

Using antibodies against pig brain phosphate activated glutaminase, the enzyme appears to be rather conservative as we have observed immuno- staining in the brain from all species investegated [pig, cow, rabbit, rat, mouse, man, fish (cod and salmon) and bird (chicken)]. In addition, phosphate activated glutaminase from cultured mouse cerebral cortex inter- neurons (mainly GABA-ergic), cerebellar granule cells (glutamatergic) and astrocytes stained in an analogous manner. However, no phosphate activated glutaminase-like immunostaining was found in lobster ganglion. yeast and E. coli. Using the Western blotting technique, phosphate activated glutaminase from dodecyl sulfate treated samples from all the above mentioned preparations revealed a MW close to 64 K_d. The MW is similar to the MW of the subunit of phosphate activated glutaminase in a highly purified pig brain preparation, The Western blotting technique seems to be well suited to identify phosphate activated glutaminase-like immunoreactivity in different tissues.     

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     

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)     

PHOSPHATE ACTIVATED GLUTAMINASE ACTIVITY AND GLUTAMINE UPTAKE IN PRIMARY CULTURES OF ASTROCYTES

Abstract— Uptake and release of glutamine were measured in primary cultures of astrocytes together with the activity of the phosphate activated glutaminase (EC 3.5.1.2). In contrast to previous findings of an effective, high affinity uptake of other amino acids (e.g. glutamate, GABA) no such uptake of glutamine was observed, though a saturable, concentrative uptake mechanism did exist (K _m = 3.3 ± 0.5 m m ; V _max= 50.2 ± 12.6 nmol ± min⁻¹± mg⁻¹). The phosphate activated glutaminase activity in the astrocytes (6.9 ± 0.9 nmol ± min⁻¹± mg⁻¹) was similar to the activity found in whole brain (5.4 ± 0.7 nmol ± min ^−l± mg⁻¹), which may contrast with previous findings of a higher activity of the glutamine synthetase (EC 6.3.1.2) in astrocytes than in whole brain. The observations are compatible with the hypothesis of an in vivo flow of glutamate (and GABA) from neurons to astrocytes where it is taken up and metabolized, and a compensatory flow of glutamine towards neurons and away from astrocytes although the latter cell type may be more deeply involved in glutamine metabolism than envisaged in the hypothesis.     

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.     

Characteristics of Triticale glutaminase]

The glutaminase (EC 3.5.1.2) isolated from seedlings of triticale (Triticale sp.) had a pH optimum of about 8, was inhibited with excess substrate (glutamine), and reaction products (glutamate and NH4+). A monovalent anion (Cl-) and a multivalent anion (phosphate) were shown to activate the glutaminase. Some features of the glutaminase from triticale were similar to those of animal glutaminase activated by phosphate and were different from features of the enzyme from Escherichia coli.     

Ontogenetic development of glutamate metabolizing enzymes in cultured cerebellar granule cells and in cerebellumin vivo

The ontogenetic development of the enzymes phosphate activated glutaminase (PAG), glutamate dehydrogenase (GLDH), glutamic-oxaloacetic-transaminase (GOT), glutamine synthetase (GS), and ornithine--aminotransferase (Orn-T) was followed in cerebellum in vivo and in cultured cerebellar granule cells. It was found that PAG, GLDH, and GOT exhibited similar developmental patterns in the cultured neurons compared to cerebellum. PAG showed, however, a more pronounced phosphate activation in the cultured granule cells compared to in vivo. The activity of GS remained low in the cultured neurons compared to the increasing activity of this enzyme found in vivo. On the other hand Orn-T exhibited an increase in its specific activity in the cultured cells as a function of time in culture in contrast to the non-changing activity of this enzyme in vivo. Compared to cerebellum the cultured neurons exhibited higher activities of GLDH, GOT, and Orn-T whereas the activity of PAG was only slightly higher in the cultured cells. The activity of GS in the cultured neurons was only 5–10% of the activity in cerebellum in vivo. It is concluded that cultured cerebellar granule cells represent a reliable model system by which the metabolism and function of glutamatergic neurons can be conveniently studied in a physiologically meaningful way.     

Is there In Vivo Evidence for Amino Acid Shuttles Carrying Ammonia from Neurons to Astrocytes?

Neurochemical Research - The high in vivo flux of the glutamate/glutamine cycle puts a strong demand on the return of ammonia released by phosphate activated glutaminase from the neurons to the...     

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.     

Response of rat liver glutaminase to pH, ammonium, and citrate. Possible regulatory role of glutaminase in ureagenesis

Liver glutaminase is stimulated by an increase in NH4+ concentration and NH4+ is an absolute requirement for activity at approximate physiological concentrations of phosphate and glutamine. Increases in the concentration of NH4+ cannot, however, overcome the inhibitory effect of a decrease in pH. In addition, the concentration of NH4+ required for half-maximal rate decreases as pH increases. This decrease is the result of two factors: a direct effect of pH on the apparent affinity of the enzyme for NH4+, and an indirect effect of pH brought about by an increase in the apparent affinity of the enzyme for phosphate which results in a further decrease in the M0.5 for NH4+. In addition, liver glutaminase responds strongly to the concentration of citrate over a physiologically relevant range at approximate physiological concentrations of NH4+, phosphate, and glutamine. An increase in citrate concentration stimulates glutaminase by increasing the affinity of the enzyme for glutamine. The apparent affinity of the enzyme for citrate increases as pH increases. The strong response of liver glutaminase to pH, NH4+, and citrate and the fact that the hydrolysis of glutamine can supply metabolites and effectors for urea synthesis suggest a possible regulatory role of glutaminase in ureagenesis.     

Characterization of Glutaminase from Triticale

The glutaminase (EC 3.5.1.2) isolated from seedlings of triticale (Triticalesp.) had a pH optimum of about 8, was inhibited with excess substrate (glutamine), and reaction products (glutamate and NH⁺ ₄). A monocharged anion (Cl^–) and a multicharged anion (phosphate) were shown to activate the glutaminase. Some features of the glutaminase from triticale were similar to those of animal glutaminase activated by phosphate and were different from features of the enzyme from Escherichia coli.     

Localization and some properties of phosphate-dependent glutaminase in disrupted liver mitochondria.

1. Glutaminase activity in frozen and thawed liver mitochondria was activated by NH4+, phosphate and HCO3-ions and also by ATP . 2. NH4+ and HCO3-ions decreased the requirement of the enzyme for phosphate. The activation by ATP was observed only in the presence of NH4+ or HCO3-ions. 3. In frozen-and-thawed mitochondria, the enzyme was loosely bound to the inner membrane, the Arrhenius plot showing a break at 23 degrees C. On sonication, glutaminase was detached from the membrane and the Arrhenius plot became linear. 4. The apparent Km for glutamine of the membrane-bound form was 6 mM, and that of the soluble form was 21 mM. 5. It is likely that the properties of glutaminase in the intact cell are dependent on the association of this enzyme with the mitochondrial membrane.     

ACTIVATION OF PIG BRAIN GLUTAMINASE

Pig brain glutaminase (EC 3.5.1.2, l -glutamine amidohydrolase) is activated by certain anions (e.g. phosphate, fluoride, carboxylic acids) and inhibited by others (e.g. chloride, bromide, iodide and glutamate). The only cation which has been found to activate the enzyme is the ammonium ion. This applies to both the tris-HCl form and the phosphate-borate form of glutaminase.     

Phosphate activated glutaminase is concentrated in mitochondria of sensory hair cells in rat inner ear: a high resolution immunogold study

Glutamate has been implicated in signal transmission between sensory hair cells and afferent fibers in the inner ear. However, the mechanisms responsible for glutamate replenishment in these cells are not known. Here we provide evidence that phosphate activated glutaminase, which is thought to be the predominant glutamate-synthesizing enzyme in the brain, is concentrated in all types of hair cell in the organ of Corti and vestibular epithelium. By use of two different antibodies (directed to the N and C terminus, respectively) it was shown that glutaminase is largely restricted to mitochondria and that part of the enzyme pool is associated with the inner membrane of this organelle. Quantitative analysis of immunogold labelled Lowicryl sections revealed that the level of glutaminase immunoreactivity in mitochondria of supporting cells is less than 15% of that in hair cell mitochondria. Using triple labelling for glutaminase, glutamate, and glutamine, evidence was provided of a positive correlation between the glutamate/glutamine ratio and the level of glutaminase immunoreactivity, suggesting that the glutaminase antibodies identify a functional enzyme pool. Our results strengthen the idea that glutamate is a hair cell transmitter and indicate that the sensory epithelia in the inner ear show a metabolic compartmentation analogous to that in the brain.     

The activity of glutaminase in the human placenta

Homogenates and extracts of human placenta are able to desamidate glutamine by means of an enzyme which has the properties of glutaminase. Placental glutaminase is activated by phosphate. Its pH optimum lies at 9.0.A method for its assay in placental homogenate is described. It was found that the glutaminase activity decreases toward the end of pregnancy. At this time, the activity, expressed as Q_NH3 (N), amounts to 23.7 ± 6.7.Some quantitative aspects of glutaminase activity in the human placenta and kidney are discussed.     

ACTIVATORS and INHIBITORS OF BRAIN GLUTAMINASE

(1) The glutaminase activity of a guinea pig brain dispersion (a 1500g supernatant solution) was tested at pH 7.5 in the presence of a series of organic acids at 20 mm with or without the further addition of 7.5 mm -phosphate. (2) In the absence of phosphate, glutaminase activity was strongly enhanced by tricarboxylic acids, less strongly by dicarboxylic acids, and slightly, if at all, by monocarboxylic acids. Acidic amino acids were intermediate between mono- and dicarboxylic acids. In the presence of 7.5 mm -phosphate, the addition of 20 mm organic acids resulted in strong potentiation of the activating effect in many cases. The activating effect of even the most active of the organic acids tested, citrate, was only about half of the effect of an equimolar amount of phosphate. (3) At phosphate concentrations approaching the saturation level for activation, the further addition of citrate was without effect. (4) Glutaminase was strongly activated by ITP which was about three times as active as inorganic phosphate. IMP was less active than inorganic phosphate and creatine phosphate had only slight activity which seemed to be accounted for by its content of inorganic phosphate. (5) Glutaminase was activated by fluoride, in the presence as well as in the absence of added phosphate. Chloride, bromide, and iodide, at 100 mm , produced increasing inhibition of the phosphate-activated reaction. The inhibiting effect of iodide was qualitatively competitive with phosphate. (6) The effects of various other potential inhibitors and activators, including SH-reagents, d -glutamine, several amino acids, and amino acid derivatives were studied. (7) The results have been discussed with particular reference to their significance in elucidating the natural function of brain glutaminase. It has been suggested that glutaminase is an allosteric enzyme and that the secondary active site requires a reaction with three anionic groups for full activation.     

Subcellular localization of rat kidney phosphate independent glutaminase

The phosphate independent glutaminase is contained in the brush border membrane of the rat kidney proximal tubule cells. This glutaminase activity cofractionates with the brush border membrane marker activities, alkaline phosphatase and γ-glutamyltranspeptidase, during differential centrifugation. About 30% of these activities are recovered with the mitochondrial fraction, the remainder is pelleted in the heavy microsomal fraction. The phosphate independent glutaminase in both fractions bands, during isopycnic centrifugation, with a mean density of 1.16–1.17 and is coincident with both brush border membrane marker activities. The isolation of intact, individual kidney cells was accomplished by initial perfusion of the kidneys in situ with a collagenase-papain solution followed by a brief incubation in the same enzyme solution. Incubation of isolated cells with a higher concentration of papain results in selective release of the phosphate independent glutaminase. The fact that this occurs without appreciable release of a cytoplasmic marker activity, lactate dehydrogenase, suggests that the phosphate independent glutaminase may be localized on the external surface of the kidney cells.     

Phosphate-Activated Glutaminase in the Crude Mitochondrial Fraction (P2 Fraction) from Human Brain Cortex

The kinetics and other properties of phosphate-activated glutaminase have for the first time been studied in the crude mitochondrial fraction (P2 fraction) from human brain. The enzyme is for unexplained reasons inactivated postmortem. The enzyme activity decreases by storing the tissue or homogenate at 37 degrees C. The inactivation is not caused by formation of a dialysable inhibiting compound. No large proteolytic degradation has occurred, since the phosphate-activated glutaminase-like immunoreactive band did not disappear during the storage. The molecular weight of the subunit of the enzyme as determined by immunoblots of sodium dodecyl sulfate-treated homogenates from human brain is estimated to be approximately 64 K. The enzyme has been shown to have a pH optimum of 8.6; it is activated by phosphate, inhibited by glutamate, and partially inhibited by ammonia. Double-inverse plots of enzyme activity against phosphate are concave-upward, and more so in the presence of an inhibitor. The inhibition by glutamate appears to be noncompetitive with the substrate glutamine, and competitive with the activator phosphate. These kinetic properties are not significantly different from our earlier observations concerning phosphate-activated glutaminase from pig brain and pig kidney.     

Valproate Increases Glutaminase and Decreases Glutamine Synthetase Activities in Primary Cultures of Rat Brain Astrocytes

Abstract: It has been proposed that hyperammonemia may be associated with valproate therapy. As astrocytes are the primary site of ammonia detoxification in brain, the effects of valproate on glutamate and glutamine metabolism in astrocytes were studied. It is well established that, because of compartmentation of glutamine synthetase, astrocytes are the site of synthesis of glutamine from glutamate and ammonia. The reverse reaction is catalyzed by the ubiquitous enzyme glutaminase, which is present in both neurons and astrocytes. In astrocytes exposed to 1.2 mM valproate, glutaminase activity increased 80% by day 2 and remained elevated at day 4; glutamine synthetase activity was decreased 30%. Direct addition of valproate to assay tubes with enzyme extracts from untreated astrocytes had significant effects only at concentrations of 10 and 20 mM, When astrocytes were exposed for 4 days to 0.3, 0.6, or 1.2 mM valproate and subsequently incubated with l -[U-¹⁴C]glutamate, label incorporation into [¹⁴C]glutamine was decreased by 11, 25, and 48%, respectively, and is consistent with a reduction in glutamine synthetase activity. Label incorporation from l -[U-¹⁴C]glutamate into [¹⁴C]aspartate also decreased with increasing concentrations of valproate. Following a 4-day exposure to 0.6 mM valproate, the glutamine levels increased 40% and the glutamate levels 100%. These effects were not directly proportional to valproate concentration, because exposure to 1.2 mM valproate resulted in a 15% decrease in glutamine levels and a 25% increase in glutamate levels compared with control cultures. Intracellular aspartate was inversely proportional to all concentrations of extracellular valproate, decreasing 60% with exposure to 1.2 mM valproate. These results indicate that valproate increases glutaminase activity, decreases glutamine synthetase activity, and alters Krebs-cycle activity in astrocytes, suggesting a possible mechanism for hyperammonemia in brain during valproate therapy.     

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.