Tumor necrosis factor-alpha induces neurotoxicity via glutamate release from hemichannels of activated microglia in an autocrine manner

Glutamate released by activated microglia induces excitoneurotoxicity and may contribute to neuronal damage in neurodegenerative diseases, including Alzheimer disease, Parkinson disease, amyotrophic lateral sclerosis, and multiple sclerosis. In addition, tumor necrosis factor-alpha (TNF-alpha) secreted from activated microglia may elicit neurodegeneration through caspase-dependent cascades and silencing cell survival signals. However, direct neurotoxicity of TNF-alpha is relatively weak, because TNF-alpha also increases production of neuroprotective factors. Accordingly, it is still controversial how TNF-alpha exerts neurotoxicity in neurodegenerative diseases. Here we have shown that TNF-alpha is the key cytokine that stimulates extensive microglial glutamate release in an autocrine manner by up-regulating glutaminase to cause excitoneurotoxicity. Further, we have demonstrated that the connexin 32 hemichannel of the gap junction is another main source of glutamate release from microglia besides glutamate transporters. Although pharmacological blockade of glutamate receptors is a promising therapeutic candidate for neurodegenerative diseases, the associated perturbation of physiological glutamate signals has severe adverse side effects. The unique mechanism of microglial glutamate release that we describe here is another potential therapeutic target. We rescued neuronal cell death in vitro by using a glutaminase inhibitor or hemichannel blockers to diminish microglial glutamate release without perturbing the physiological glutamate level. These drugs may give us a new therapeutic strategy against neurodegenerative diseases with minimum adverse side effects.     

Small molecule glutaminase inhibitors block glutamate release from stimulated microglia

Glutaminase plays a critical role in the generation of glutamate, a key excitatory neurotransmitter in the CNS. Excess glutamate release from activated macrophages and microglia correlates with upregulated glutaminase suggesting a pathogenic role for glutaminase. Both glutaminase siRNA and small molecule inhibitors have been shown to decrease excess glutamate and provide neuroprotection in multiple models of disease, including HIV-associated dementia (HAD), multiple sclerosis and ischemia. Consequently, inhibition of glutaminase could be of interest for treatment of these diseases. Bis-2-(5-phenylacetimido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES) and 6-diazo-5-oxo-l-norleucine (DON), two most commonly used glutaminase inhibitors, are either poorly soluble or non-specific. Recently, several new BPTES analogs with improved physicochemical properties were reported. To evaluate these new inhibitors, we established a cell-based microglial activation assay measuring glutamate release. Microglia-mediated glutamate levels were significantly augmented by tumor necrosis factor (TNF)-α, phorbol 12-myristate 13-acetate (PMA) and Toll-like receptor (TLR) ligands coincident with increased glutaminase activity. While several potent glutaminase inhibitors abrogated the increase in glutamate, a structurally related analog devoid of glutaminase activity was unable to block the increase. In the absence of glutamine, glutamate levels were significantly attenuated. These data suggest that the in vitro microglia assay may be a useful tool in developing glutaminase inhibitors of therapeutic interest.     

Glutaminase-containing microvesicles from HIV-1-infected macrophages and immune-activated microglia induce neurotoxicity

Background

HIV-1-infected and/or immune-activated microglia and macrophages are pivotal in the pathogenesis of HIV-1-associated neurocognitive disorders (HAND). Glutaminase, a metabolic enzyme that facilitates glutamate generation, is upregulated and may play a pathogenic role in HAND. Our previous studies have demonstrated that glutaminase is released to the extracellular fluid during HIV-1 infection and neuroinflammation. However, key molecular mechanisms that regulate glutaminase release remain unknown. Recent advances in understanding intercellular trafficking have identified microvesicles (MVs) as a novel means of shedding cellular contents. We posit that during HIV-1 infection and immune activation, microvesicles may mediate glutaminase release, generating excessive and neurotoxic levels of glutamate.

Results

MVs isolated through differential centrifugation from cell-free supernatants of monocyte-derived macrophages (MDM) and BV2 microglia cell lines were first confirmed in electron microscopy and immunoblotting. As expected, we found elevated number of MVs, glutaminase immunoreactivities, as well as glutaminase enzyme activity in the supernatants of HIV-1 infected MDM and lipopolysaccharide (LPS)-activated microglia when compared with controls. The elevated glutaminase was blocked by GW4869, a neutral sphingomyelinase inhibitor known to inhibit MVs release, suggesting a critical role of MVs in mediating glutaminase release. More importantly, MVs from HIV-1-infected MDM and LPS-activated microglia induced significant neuronal injury in rat cortical neuron cultures. The MV neurotoxicity was blocked by a glutaminase inhibitor or GW4869, suggesting that the neurotoxic potential of HIV-1-infected MDM and LPS-activated microglia is dependent on the glutaminase-containing MVs.

Conclusions

These findings support MVs as a potential pathway/mechanism of excessive glutamate generation and neurotoxicity in HAND and therefore MVs may serve as a novel therapeutic target.

     

In vitro glutaminase regulation and mechanisms of glutamate generation in HIV-1-infected macrophage

Mononuclear phagocyte (MP, macrophages and microglia) dysfunction plays a significant role in the pathogenesis of HIV-1-associated dementia (HAD) through the production and release of soluble neurotoxic factors including glutamate. Glutamate production is greatly increased following HIV-1 infection of cultured MP, a process dependent upon the glutamate-generating enzyme glutaminase. Glutaminase inhibition was previously found to significantly decrease macrophage-mediated neurotoxicity. Potential mechanisms of glutaminase-mediated excitotoxicity including enzyme up-regulation, increased enzyme activity and glutaminase localization were investigated in this report. RNA and protein analysis of HIV-infected human primary macrophage revealed up-regulation of the glutaminase isoform GAC, yet identified no changes in the kidney-type glutaminase isoform over the course of infection. Glutaminase is a mitochondrial protein, but was found to be released into the cytosol and extracellular space following infection. This released enzyme is capable of rapidly converting the abundant extracellular amino acid glutamine into excitotoxic levels of glutamate in an energetically favorable process. These findings support glutaminase as a potential component of the HAD pathogenic process and identify a possible therapeutic avenue for the treatment of neuroinflammatory states such as HAD.     

Blockade of gap junction hemichannel suppresses disease progression in mouse models of amyotrophic lateral sclerosis and Alzheimer's disease

Background

Glutamate released by activated microglia induces excitotoxic neuronal death, which likely contributes to non-cell autonomous neuronal death in neurodegenerative diseases, including amyotrophic lateral sclerosis and Alzheimer''s disease. Although both blockade of glutamate receptors and inhibition of microglial activation are the therapeutic candidates for these neurodegenerative diseases, glutamate receptor blockers also perturbed physiological and essential glutamate signals, and inhibitors of microglial activation suppressed both neurotoxic/neuroprotective roles of microglia and hardly affected disease progression. We previously demonstrated that activated microglia release a large amount of glutamate specifically through gap junction hemichannel. Hence, blockade of gap junction hemichannel may be potentially beneficial in treatment of neurodegenerative diseases.

Methods and Findings

In this study, we generated a novel blood-brain barrier permeable gap junction hemichannel blocker based on glycyrrhetinic acid. We found that pharmacologic blockade of gap junction hemichannel inhibited excessive glutamate release from activated microglia in vitro and in vivo without producing notable toxicity. Blocking gap junction hemichannel significantly suppressed neuronal loss of the spinal cord and extended survival in transgenic mice carrying human superoxide dismutase 1 with G93A or G37R mutation as an amyotrophic lateral sclerosis mouse model. Moreover, blockade of gap junction hemichannel also significantly improved memory impairments without altering amyloid β deposition in double transgenic mice expressing human amyloid precursor protein with K595N and M596L mutations and presenilin 1 with A264E mutation as an Alzheimer''s disease mouse model.

Conclusions

Our results suggest that gap junction hemichannel blockers may represent a new therapeutic strategy to target neurotoxic microglia specifically and prevent microglia-mediated neuronal death in various neurodegenerative diseases.     

Glutamate production by HIV-1 infected human macrophage is blocked by the inhibition of glutaminase

Mononuclear phagocyte (macrophages and microglia) dysfunction plays a significant role in the pathogenesis of human immunodeficiency virus (HIV) associated dementia (HAD) through the production and release of soluble neurotoxic factors including glutamate. The mechanism of glutamate regulation by HIV-1 infection remains unclear. In this report, we investigated whether the enzyme glutaminase is responsible for glutamate generation by HIV-1 infected monocyte-derived macrophages. We tested the functionality of novel small molecule inhibitors designed to specifically block the activity of glutaminase. Glutaminase inhibitors were first characterized in a kinetic assay with crude glutaminase from rat brain revealing an uncompetitive mechanism of inhibition. The inhibitors were then tested in vitro for their ability to prevent glutamate generation by HIV-infected macrophages, their effect upon macrophage viability, and HIV infection. To validate these findings, glutaminase specific siRNA was tested for its ability to prevent glutamate increase during infection. Our results show that both glutaminase specific small molecule inhibitors and glutaminase specific siRNA were effective at preventing increases in glutamate by HIV-1 infected macrophage. These findings support glutaminase as a potential component of the HAD pathogenic process and identify a possible therapeutic avenue for the treatment of neuroinflammatory states such as HAD.     

Neurotoxicity of microglial cathepsin D revealed by secretome analysis

Microglia-driven inflammatory responses have both neuroprotective and neurotoxic effects in the CNS. The excessive and chronic activation of microglia, however, may shift the balance towards neurotoxic effects. In this regard, proteins secreted from activated microglia likely play a key role in the neurotoxic effects. To characterize secreted proteins of activated microglia, conditioned media obtained from BV-2 mouse microglia cells were analyzed by two-dimensional gel electrophoresis or liquid chromatography coupled with tandem mass spectrometry. Among many proteins identified in the secretome of activated microglia, an aspartic endoprotease cathepsin D has been found to mediate microglial neurotoxicity based on the following results: (i) the expression of cathepsin D protein was markedly increased in lipopolysaccharide/interferon-γ-stimulated microglia compared with resting microglia as determined by western blot analysis of conditioned media; (ii) knockdown of cathepsin D expression in microglia using short hairpin RNA diminished the neurotoxicity in the coculture of microglia and neuroblastoma cells and (iii) recombinant procathepsin D protein exerted cytotoxic effects toward cultured neurons. In conclusion, cathepsin D appears to play a central role in the microglial neurotoxicity, and could be a potential biomarker or drug target for the diagnosis and treatment of neurodegenerative diseases that are associated with excessive microglial activation and subsequent neurotoxic inflammation.     

Prominent glutamine oxidation activity in mitochondria of avian transplantable hepatoma induced by MC-29 virus

Well coupled mitochondria were isolated from transplantable chicken hepatoma induced by MC-29 virus. The mitochondrial phosphate-dependent and phosphate-independent glutaminase activities were increased compared with those from normal chicken liver. Glutamate dehydrogenase was undetectable in the tumor mitochondria. Oxypolarographic tests showed the following: glutamine oxidation was prominent in the tumor mitochondria and was mediated through an NAD-linked reaction, while mitochondria from the liver showed a feeble glutamine oxidation; glutamine oxidation by tumor mitochondria was inhibited either by aminooxyacetate, inhibitor of transaminases, or prior incubation of mitochondria with DON (6-diazo-5-oxonorleucine), which inhibited mitochondrial glutaminases. Bromofuroate, inhibitor of glutamate dehydrogenase, had little or no effect; and glutamate oxidation was also inhibited by aminooxyacetate, while it was not affected by DON. These findings clearly show a high glutamate oxidation activity in the hepatoma and indicate that the product of glutamine hydrolysis, glutamate, is catabolized via transamination in the mitochondria to supply ATP.     

Kinetic characterization of ebselen,chelerythrine and apomorphine as glutaminase inhibitors

Glutaminase catalyzes the hydrolysis of glutamine to glutamate and plays a central role in the proliferation of neoplastic cells via glutaminolysis, as well as in the generation of excitotoxic glutamate in central nervous system disorders such as HIV-associated dementia (HAD) and multiple sclerosis. Both glutaminase siRNA and glutaminase inhibition have been shown to be effective in in vitro models of cancer and HAD, suggesting a potential role for small molecule glutaminase inhibitors. However, there are no potent, selective inhibitors of glutaminase currently available. The two prototypical glutaminase inhibitors, BPTES and DON, are either insoluble or non-specific. In a search for more drug-like glutaminase inhibitors, we conducted a screen of 1280 in vivo active drugs (Library of Pharmacologically Active Compounds (LOPAC¹²⁸⁰)) and identified ebselen, chelerythrine and (R)-apomorphine. The newly identified inhibitors exhibited 10 to 1500-fold greater affinities than DON and BPTES and over 100-fold increased efficiency of inhibition. Although non-selective, it is noteworthy that the affinity of ebselen for glutaminase is more potent than any other activity yet described. It is possible that the previously reported biological activity seen with these compounds is due, in part, to glutaminase inhibition. Ebselen, chelerythrine and apomorphine complement the armamentarium of compounds to explore the role of glutaminase in disease.     

HIV-infected macrophages mediate neuronal apoptosis through mitochondrial glutaminase

A significant number of patients infected with human immunodeficiency virus-1 (HIV-1) suffer cognitive impairment ranging from mild to severe HIV-associated dementia (HAD), a result of neuronal degeneration in the basal ganglia, cerebral cortex and hippocampus. Mononuclear phagocyte dysfunction is thought to play an important role in the pathogenesis of HAD. Glutamate neurotoxicity is triggered primarily by massive Ca²⁺ influx arising from over-stimulation of the NMDA subtype of glutamate receptors. The underlying mechanisms, however, remain elusive. We have tested the hypothesis that mitochondrial glutaminase in HIV-infected macrophages is involved in converting glutamine to glutamate. Our results demonstrate that the concentration of glutamate in HIV-1 infected conditioned media was dependent on glutamine dose, and HIV-1 infected conditioned medium mediated glutamine-dependent neurotoxicity. These results indicate HIV-infection mediates neurotoxicity through glutamate production. In addition, glutamate-mediated neurotoxicity correlated with caspase activation and neuronal cell cycle re-activation. Inhibition of mitochondrial glutaminase diminished the HIV-induced glutamate production, and attenuated NMDA over-stimulation and subsequent neuronal apoptosis. These data implicate mitochondrial glutaminase in the induction of glutamate-mediated neuronal apoptosis during HIV-associated dementia, and provides a possible therapeutic strategy for HAD treatment.     

Activation of microglia by secreted amyloid precursor protein evokes release of glutamate by cystine exchange and attenuates synaptic function

Microglial activation as part of a chronic inflammatory response is a prominent component of Alzheimer's disease. Secreted forms of the beta-amyloid precursor protein (sAPP) previously were found to activate microglia, elevating their neurotoxic potential. To explore neurotoxic mechanisms, we analyzed microglia-conditioned medium for agents that could activate glutamate receptors. Conditioned medium from primary rat microglia activated by sAPP caused a calcium elevation in hippocampal neurons, whereas medium from untreated microglia did not. This response was sensitive to the NMDA receptor antagonist, aminophosphonovaleric acid. Analysis of microglia-conditioned by HPLC revealed dramatically higher concentrations of glutamate in cultures exposed to sAPP. Indeed, the glutamate levels in sAPP-treated cultures were substantially higher than those in cultures treated with amyloid beta-peptide. This sAPP-evoked glutamate release was completely blocked by inhibition of the cystine-glutamate antiporter by alpha-aminoadipate or use of cystine-free medium. Furthermore, a sublethal concentration of sAPP compromised synaptic density in microglia-neuron cocultures, as evidenced by neuronal connectivity assay. Finally, the neurotoxicity evoked by sAPP in microglia-neuron cocultures was attenuated by inhibitors of either the neuronal nitric oxide synthase (N(G)-propyl-L-arginine) or inducible nitric oxide synthase (1400 W). Together, these data indicate a scenario by which microglia activated by sAPP release excitotoxic levels of glutamate, probably as a consequence of autoprotective antioxidant glutathione production within the microglia, ultimately causing synaptic degeneration and neuronal death.     

Heat induction of heat shock protein 25 requires cellular glutamine in intestinal epithelial cells

Glutamine is considered a nonessential amino acid; however, it becomes conditionally essential during critical illness when consumption exceeds production. Glutamine may modulate the heat shock/stress response, an important adaptive cellular response for survival. Glutamine increases heat induction of heat shock protein (Hsp) 25 in both intestinal epithelial cells (IEC-18) and mesenchymal NIH/3T3 cells, an effect that is neither glucose nor serum dependent. Neither arginine, histidine, proline, leucine, asparagine, nor tyrosine acts as physiological substitutes for glutamine for heat induction of Hsp25. The lack of effect of these amino acids was not caused by deficient transport, although some amino acids, including glutamate (a major direct metabolite of glutamine), were transported poorly by IEC-18 cells. Glutamate uptake could be augmented in a concentration- and time-dependent manner by increasing either media concentration and/or duration of exposure. Under these conditions, glutamate promoted heat induction of Hsp25, albeit not as efficiently as glutamine. Further evidence for the role of glutamine conversion to glutamate was obtained with the glutaminase inhibitor 6-diazo-5-oxo-L-norleucine (DON), which inhibited the effect of glutamine on heat-induced Hsp25. DON inhibited phosphate-dependent glutaminase by 75% after 3 h, decreasing cell glutamate. Increased glutamine/glutamate conversion to glutathione was not involved, since the glutathione synthesis inhibitor, buthionine sulfoximine, did not block glutamine’s effect on heat induction of Hsp25. A large drop in ATP levels did not appear to account for the diminished Hsp25 induction during glutamine deficiency. In summary, glutamine is an important amino acid, and its requirement for heat-induced Hsp25 supports a role for glutamine supplementation to optimize cellular responses to pathophysiological stress. IEC-18; NIH/3T3; glutaminase; 6-diazo-5-oxo-L-norleucine; glutathione     

IL‐1β and TNF‐α induce neurotoxicity through glutamate production: a potential role for neuronal glutaminase

Glutaminase 1 is the main enzyme responsible for glutamate production in mammalian cells. The roles of macrophage and microglia glutaminases in brain injury, infection, and inflammation are well documented. However, little is known about the regulation of neuronal glutaminase, despite neurons being a predominant cell type of glutaminase expression. Using primary rat and human neuronal cultures, we confirmed that interleukin‐1β (IL‐1β) and tumor necrosis factor‐α (TNF‐α), two pro‐inflammatory cytokines that are typically elevated in neurodegenerative disease states, induced neuronal death and apoptosis in vitro. Furthermore, both intracellular and extracellular glutamate levels were significantly elevated following IL‐1β and/or TNF‐α treatment. Pre‐treatment with N‐Methyl‐d ‐aspartate (NMDA) receptor antagonist MK‐801 blocked cytokine‐induced glutamate production and alleviated the neurotoxicity, indicating that IL‐1β and/or TNF‐α induce neurotoxicity through glutamate. To determine the potential source of excess glutamate production in the culture during inflammation, we investigated the neuronal glutaminase and found that treatment with IL‐1β or TNF‐α significantly upregulated the kidney‐type glutaminase (KGA), a glutaminase 1 isoform, in primary human neurons. The up‐regulation of neuronal glutaminase was also demonstrated in situ in a murine model of HIV‐1 encephalitis. In addition, IL‐1β or TNF‐α treatment increased the levels of KGA in cytosol and TNF‐α specifically increased KGA levels in the extracellular fluid, away from its main residence in mitochondria. Together, these findings support neuronal glutaminase as a potential component of neurotoxicity during inflammation and that modulation of glutaminase may provide therapeutic avenues for neurodegenerative diseases.     

Macrophages treated with particulate matter PM2.5 induce selective neurotoxicity through glutaminase‐mediated glutamate generation

Exposure to atmospheric particulate matter PM_2.5 (aerodynamic diameter ≤ 2.5 μm) has been epidemiologically associated with respiratory illnesses. However, recent data have suggested that PM_2.5 is able to infiltrate into circulation and elicit a systemic inflammatory response. Potential adverse effects of air pollutants to the central nervous system (CNS) have raised concerns, but whether PM_2.5 causes neurotoxicity remains unclear. In this study, we have demonstrated that PM_2.5 impairs the tight junction of endothelial cells and increases permeability and monocyte transmigration across endothelial monolayer in vitro, indicating that PM_2.5 is able to disrupt blood–brain barrier integrity and gain access to the CNS. Exposure of primary neuronal cultures to PM_2.5 resulted in decrease in cell viability and loss of neuronal antigens. Furthermore, supernatants collected from PM_2.5‐treated macrophages and microglia were also neurotoxic. These macrophages and microglia significantly increased extracellular levels of glutamate following PM_2.5 exposure, which were negatively correlated with neuronal viability. Pre‐treatment with NMDA receptor antagonist MK801 alleviated neuron loss, suggesting that PM_2.5 neurotoxicity is mediated by glutamate. To determine the potential source of excess glutamate production, we investigated glutaminase, the main enzyme for glutamate generation. Glutaminase was reduced in PM_2.5‐treated macrophages and increased in extracellular vesicles, suggesting that PM_2.5 induces glutaminase release through extracellular vesicles. In conclusion, these findings indicate PM_2.5 as a potential neurotoxic factor, crucial to understanding the effects of air pollution on the CNS.

     

Mitochondrial glutaminase enhances extracellular glutamate production in HIV-1-infected macrophages: linkage to HIV-1 associated dementia

Dysfunction in mononuclear phagocyte (MP, macrophages and microglia) immunity is thought to play a significant role in the pathogenesis of HIV-1 associated dementia (HAD). In particular, elevated extracellular concentrations of the excitatory neurotransmitter glutamate, produced by MP as a consequence of viral infection and immune activation, can induce neuronal injury. To determine the mechanism by which MP-mediated neuronal injury occurs, the concentration and rates of production of extracellular glutamate were measured in human monocyte-derived macrophage (MDM) supernatants by reverse phase high-performance liquid chromatography (RP-HPLC). Measurements were taken of supernatants from MDM infected with multiple HIV-1 strains including ADA and DJV (macrophage tropic, M-tropic), and 89.6 (dual tropic). High levels of glutamate were produced by MDM infected with M-tropic viruses. AZT, an inhibitor of HIV-1 replication, inhibited glutamate generation, demonstrating a linkage between HIV-1 infection and enhanced glutamate production. In our culture system, glutamate production was dependent upon the presence of glutamine and was inhibited by 6-diazo-5-oxo-L-norleucine, a glutaminase inhibitor. Supernatants collected from HIV-1-infected MP generated more glutamate following glutamine addition than supernatants isolated from uninfected MP. These findings implicate the involvement of a glutamate-generating enzyme, such as phosphate-activated mitochondrial glutaminase (PMG) in MP-mediated glutamate production.     

Activation of group II metabotropic glutamate receptors underlies microglial reactivity and neurotoxicity following stimulation with chromogranin A,a peptide up-regulated in Alzheimer's disease

Regulation of microglial reactivity and neurotoxicity is critical for neuroprotection in neurodegenerative diseases. Here we report that microglia possess functional group II metabotropic glutamate receptors, expressing mRNA and receptor protein for mGlu2 and mGlu3, negatively coupled to adenylate cyclase. Two different agonists of these receptors were able to induce a neurotoxic microglial phenotype which was attenuated by a specific antagonist. Chromogranin A, a secretory peptide expressed in amyloid plaques in Alzheimer's disease, activates microglia to a reactive neurotoxic phenotype. Chromogranin A-induced microglial activation and subsequent neurotoxicity may also involve an underlying stimulation of group II metabotropic glutamate receptors since their inhibition reduced chromogranin A-induced microglial reactivity and neurotoxicity. These results show that selective inhibition of microglial group II metabotropic glutamate receptors has a positive impact on neuronal survival, and may prove a therapeutic target in Alzheimer's disease.     

p-aminobenzoate synthesis in Escherichia coli: kinetic and mechanistic characterization of the amidotransferase PabA.

p-Aminobenzoic acid (PABA) is an important precursor in the bacterial biosynthetic pathway for folate enzymes. This biosynthesis requires three separate proteins: PabA, PabB, and PabC. Together PabA and PabB convert glutamine and chorismate to glutamate and 4-amino-4-deoxychorismate. This aminochorismate is subsequently transformed to PABA by PabC. In this study, PabA from Escherichia coli has been purified to homogeneity from an overproducing construct and found to have no detectable glutaminase activity until addition of the E. coli PabB subunit. PabB forms a 1:1 complex with PabA to yield a glutaminase k(cat) of 17 min-1. The addition of chorismate, the substrate of PabB, induces a 2-fold increase of k(cat) as well as a 3-fold increase of Km for glutamine. The PabA/PabB complex has Kd less than 10(-8) M but does not form a stable complex isolable by gel filtration. Studies with the glutamine affinity label diazooxonorleucine (DON) reveal it is an inactivator of the glutaminase activity of the PabA/PabB complex, but DON does not alkylate and inactivate PabA alone. Similarly, while isolated PabA shows no tendency to form a glutamyl-enzyme intermediate, the PabA/PabB complex forms a covalent intermediate with [14C]glutamine on PabA that accumulates to 0.56 mol/mol in hydrolytic turnover. PabA is thus a conditional glutaminase, activated by 1:1 complexation with PabB.     

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