Regulation of glutaminase B in Escherichia coli. I. Purification, properties, and cold lability.

Escherichia coli contains two glutaminases, A and B, with pH optima below pH 5 and above pH 7, respectively. Neither glutaminase A nor B is released from E. coli by osmotic shock. Glutaminase B has been purified 6,000-fold and the purified preparation is estimated to contain about 40% glutaminase B. The enzyme has a molecular weight of 90,000 and an isoelectric point of 5.4. Glutaminase B exhibits a broad pH optimum between 7.1 and 9.0. Only L-glutamine is deamidated by glutaminase B, L-asparagine and D-glutamine are not deamidated. The substrate saturation curve for glutaminase B shows an intermediary plateau region. Like many regulatory enzymes, glutaminase B is cold-labile. The enzyme is inactivated by cooling and activated by warming; both processes are first order with respect to time. The activation energy for activation by warming was calculated to be 5900 cal/mol. Activation by warming increased the Vmax and decreased the S0.5 for L-glutamine, but did not alter the molecular weight of the catalytically active enzyme. Borate and glutamate protected glutaminase B from inactivation by cold.     

LOCALIZATION OF GLUTAMINASE IN THE RAT NEOSTRIATUM

Abstract— Glutaminase activity in rat striatal tissue was not significantly decreased by lesions of the cortico-striatal tract which depressed striatal glutamate uptake by 47% but was markedly decreased following intrastriatal injections of kainic acid. There also appeared to be a linear correlation between glutaminase and glutamic acid decarboxylase activities in the substantia nigra of rats injected intrastriatally with kainic acid. The results suggest that most of the glutaminase activity in these regions is localized in GABAergic structures and provide no evidence for the occurrence of this enzyme in nerve endings of the glutamergic cortico-striatal tract.     

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.     

The role of glutaminase in the small intestine

Glutaminase is the enzyme which hydrolyses glutamine, the main respiratory fuel of the intestine, to yield glutamate and ammonia. Glutaminase has a central role in intestinal metabolism: the products of the reaction catalyzed by glutaminase can be transaminated, catabolized to yield energy or used for the biosynthesis of pyrimidine nucleotides. Experimental treatments which deprive the intestine of glutamine induce intestinal atrophy. In this review, attention is paid to the role of glutaminase in intestinal metabolism. Background information on the structure, kinetics and distribution of glutaminase precede a discussion of the metabolism of glutamine within the intestine. In closing, we review the factors known to regulate glutaminase activity and emphasise that the regulation of glutaminase within the intestine is poorly understood.     

Pyruvate Carboxylase Deficiency in Pleiotropic Carbohydrate-Negative Mutant Strains of Pseudomonas aeruginosa

Cell extracts of Pseudomonas aeruginosa strain PAO were found to contain pyruvate carboxylase activity. Specific activities were minimal when cells were grown on Casamino Acids, acetate, or succinate, but were three- to fourfold higher when cells were grown in glucose, gluconate, glycerol, lactate, or pyruvate minimal media. The reaction in crude cell extracts and in partially purified preparations was dependent on pyruvate, adenosine 5'-triphosphate, and Mg(2+), but was not affected by either the presence or absence of acetyl coenzyme A. Activity was nearly totally inhibited by avidin and this inhibition was substantially blocked by free biotin in incubation mixtures. Cell extracts were shown to fix (14)CO(2) in a reaction that had these same characteristics. Eight pleiotropic, carbohydrate-negative mutant strains of the organism were isolated after nitrosoguanidine mutagenesis. Each mutant strain grew normally in acetate, succinate, and citrate minimal media but failed to utilize glucose, gluconate, 2-ketogluconate, mannitol, glycerol, lactate, and pyruvate as sole sources of carbon and energy. These strains were found by quantitative transductional analysis with phage F116 to form a single linkage group. Cell extracts of each mutant strain were either lacking or severely deficient in pyruvate carboxylase activity. Spontaneous revertants of five of the eight strains were isolated and found to recover simultaneously both pyruvate carboxylase activity and the ability to utilize each of the C(6) and C(3) compounds. A second linkage group of similar mutant strains that grew on the C(3) compounds was found to contain normal levels of pyruvate carboxylase activity, but each strain was deficient in an enzyme of the Entner-Doudoroff pathway.     

Role of glutamine aminotransferase in glutamine catabolism by Saccharomyces cerevisiae under microaerophilic conditions

The involvement of glutamine aminotransferase activity in glutamine catabolism by Saccharomyces cerevisiae under microaerophilic conditions was studied. We were able to show that there are at least two different glutamine aminotransferase activities that are differentiated genetically, by their substrate specificity (pyruvate and glyoxylate dependence), and their different modes of regulation. The pyruvate-dependent glutamine aminotransferase activity plays a major role in glutamine catabolism under microaerophilic conditions since the wild-type strain S288C showed a 10-fold higher activity in static cultures than in agitated ones. The same strain also had 3-fold higher glutaminase B activity in agitated cultures than in static ones. Pyruvate-dependent glutamine aminotransferase activity is not regulated directly by O2 itself since a rho- strain showed a high activity regardless of the extent of aeration of cultures. Finally, we were able to isolate a mutant, strain CN20, derived from the rho- strain and unable to utilize glutamine as the sole nitrogen source, which was severely affected in pyruvate-dependent but not in glyoxylate-dependent aminotransferase activity.     

Crystal structure of a major fragment of the salt-tolerant glutaminase from Micrococcus luteus K-3

Glutaminase of Micrococcus luteus K-3 (intact glutaminase; 48kDa) is digested to a C-terminally truncated fragment (glutaminase fragment; 42kDa) that shows higher salt tolerance than that of the intact glutaminase. The crystal structure of the glutaminase fragment was determined at 2.4A resolution using multiple-wavelength anomalous dispersion (MAD). The glutaminase fragment is composed of N-terminal and C-terminal domains, and a putative catalytic serine-lysine dyad (S64 and K67) is located in a cleft of the N-terminal domain. Mutations of the S64 or K67 residues abolished the enzyme activity. The N-terminal domain has abundant glutamic acid residues on its surface, which may explain its salt-tolerant mechanism. A diffraction analysis of the intact glutaminase crystals (a twinning fraction of 0.43) located the glutaminase fragment in the unit cell but failed to turn up clear densities for the missing C-terminal portion of the molecule.     

Manganese is required for oxidative metabolism in unstressed Bradyrhizobium japonicum cells

Recent studies of Mn(2+) transport mutants indicate that manganese is essential for unstressed growth in some bacterial species, but is required primarily for induced stress responses in others. A Bradyrhizobium japonicum mutant defective in the high-affinity Mn(2+) transporter gene mntH has a severe growth phenotype under manganese limitation, suggesting a requirement for the metal under unstressed growth. Here, we found that activities of superoxide dismutase and the glycolytic enzyme pyruvate kinase were deficient in an mntH strain grown under manganese limitation. We identified pykM as the only pyruvate kinase-encoding gene based on deficiency in activity of a pykM mutant, rescue of the growth phenotype with pyruvate, and pyruvate kinase activity of purified recombinant PykM. PykM is unusual in that it required Mn(2+) rather than Mg(2+) for high activity, and that neither fructose-1,6-bisphosphate nor AMP was a positive allosteric effector. The mntH-dependent superoxide dismutase is encoded by sodM, the only expressed superoxide dismutase-encoding gene under unstressed growth conditions. An mntH mutant grew more slowly on pyruvate under manganese-limited conditions than did a pykM sodM double mutant, implying additional manganese-dependent processes. The findings implicate roles for manganese in key steps in unstressed oxidative metabolism in B. japonicum.     

Glutaminase in neurons and astrocytes cultured from mouse brain: Kinetic properties and effects of phosphate,glutamate, and ammonia

Phosphate activated glutaminase comprises two kinetically distinguishable enzyme forms in cultures of cerebellar granule cells, of cortical neurons and of astrocytes. Specific activity of glutaminase is higher in cultured neurons compared with astrocytes. Glutaminase is activated by phosphate in all cell types investigated, however, glutaminase in astrocytes reguires a much higher concentration of phosphate for half maximal activation. One of the products, glutamate, inhibits the enzyme strongly, whereas the other product ammonia has only a slight inhibitory action on the enzyme.     

Isolation and characterization of salt-tolerant glutaminases from marine Micrococcus luteus K-3

Marine Micrococcus luteus K-3 constitutively produced two salt-tolerant glutaminases, designated glutaminase I and II. Glutaminase I was homogeneously purified about approximately, 1620-fold with a 4% yield, and was a dimer with a molecular weight of about 86,000. Glutaminase II was partially purified about 190-fold with a 0.04% yield. The molecular weight of glutaminase II was also 86,000. Maximum activity of glutaminase I was observed at pH 8.0, 50°C and 8–16% NaCl. The optimal pH and temperature of glutaminase II were 8.5 and 50°C. The activity of glutaminase II was not affected by the presence of 8 to 16% NaCl. The presence of 10% NaCl enhanced thermal stability of glutaminase I. Both enzymes catalyzed the hydrolysis of l-glutamine, but not its hydroxylaminolysis. The K_m values for l-glutamine were 4.4 (glutaminase I) and 6.5 mM (glutaminase II). Neither of the glutaminases were activated by the addition of 2 mM phosphate or 2 mM sulfate. p-Chloromercuribenzoate (0.01 mM) significantly inhibited glutaminase I, but not glutaminase II. The conserved sequences LA**V and V**GGT*A were observed in the N-terminal amino acid sequences of glutaminase I, similar to that for other glutaminases.     

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.     

Regulation of flux through glutaminase and glutamine synthetase in isolated perfused rat liver

1. Glutaminase and glutamine synthetase are simultaneously active in the intact liver, resulting in an energy consuming cycling of glutamine at a rate up to 0.2 mumol per g per min. 2. An increase in portal glutamine concentration was followed by an increased flux through glutaminase, but flux through glutamine synthetase remained unchanged. Glutaminase flux was also increased by ammonium ions or glucagon; these effects were additive. 3. Glutamine synthetase flux was increased by ammonium ions, but this activation was partly overcome by increasing portal glutamine concentrations. Glutamine synthetase flux was slightly increased by glucagon at portal glutamine concentrations of about 0.2-0.3 mM, but was strongly inhibited above 0.6 mMs. 4. During experimental metabolic acidosis there was an increased net release of glutamine by the liver, being due to opposing changes of flux through glutaminase and glutamine synthetase. Conversely, an increased glutamine uptake by the liver during metabolic alkalosis was observed due to an inhibition of glutamine synthetase and an activation of glutaminase. However, the two enzyme activities respond differently depending on whether glucagon or ammonium ions are present.     

Mechanism of l-Glutamine Production by an l-Glutamine-Producing Mutant of Flavobacterium rigense

Properties of some enzymes involved in l-glutamine biosynthesis in an l-glutamine-producing mutant of Flavobacterium rigense were examined. Glutamate-oxaloacetate transaminase in the mutant was nearly at the same level as that in the parent strain and was the most active among the enzymes participating in glutamate biosynthesis from alpha-ketoglutarate. Glutamine synthetase formation in the mutant was enhanced by increasing the concentration of (NH(4))(2)-fumarate in the medium, but the activity of this enzyme in the parent strain was very low, and its formation was not influenced by the concentration of (NH(4))(2)-fumarate. Glutaminase formation by both strains was similar and was not influenced by the levels of (NH(4))(2)-fumarate. Glutaminase activity of the mutant was inhibited by ammonia and fumarate. Intracellular amino acids and extracellular free amino acids in the mutant were compared with those of the parent strain. It seems reasonable to conclude that l-glutamine leaks out specifically through the cell membrane of strain 703 and that this specific excretion of l-glutamine probably allows a continuous conversion of l-glutamate to l-glutamine inside the cell.     

Glutamate synthase. Properties of the glutamine-dependent activity.

Properties of glutamine-dependent glutamate synthase have been investigated using homogeneous enzyme from Escherichia coli K-12. In contrast to results with enzyme from E. coli strain B (Miller, R. E., and Stadtman, E. R. (1972) J. Biol. Chem. 247, 7407-7419), this enzyme catalyzes NH3-dependent glutamate synthase activity. Selective inactivation of glutamine-dependent activity was obtained by treatment with the glutamine analog. L-2-amino-4-oxo-5-chloropentanoic acid (chloroketone). Inactivation by chloroketone exhibited saturation kinetics; glutamine reduced the rate of inactivation and exhibited competitive kinetics. Iodoacetamide, other alpha-halocarbonyl compounds, and sulfhydryl reagents gave similar selective inactivation of glutamine-dependent activity. Saturation kinetics were not obtained for inactivation by iodoacetamide but protection by glutamine exhibited competitive kinetics. The stoichiometry for alkylation by chloroketone and iodoacetamide was approximately 1 residue per protomer of molecular weight approximately 188,000. The single residue alkylated with iodo [1-14C]acetamide was identified as cysteine by isolation of S-carboxymethylcysteine. This active site cysteine is in the large subunit of molecular weight approximately 153,000. The active site cysteine was sensitive to oxidation by H2O2 generated by autooxidation of reduced flavin and resulted in selective inactivation of glutamine-dependent enzyme activity. Similar to other glutamine amidotransferases, glutamate synthase exhibits glutaminase activity. Glutaminase activity is dependent upon the functional integrity of the active site cysteine but is not wholly dependent upon the flavin and non-heme iron. Collectively, these results demonstrate that glutamate synthase is similar to other glutamine amidotransferases with respect to distinct sites for glutamine and NH3 utilization and in the obligatory function of an active site cysteine residue for glutamine utilization.     

Functional significance of the activities of glutaminase and ornithine-ω-aminotransferase in rat brain

The activities of glutaminase, glutamine synthetase (GS), arginase and ornithine amino transferase (orn-T) were studied in three regions of rat brain in heightened neuronal activity by producing convulsions by leptazol. These enzymes were studied in preconvulsive, convulsive and postconvulsive phases. Glutaminase activity was found to increase in all the three regions in the preconvulsive and convulsive phases. GS activity decreased in the preconvulsive phase but rose gradually to the control level when the postconvulsive phase was reached. The activity of arginase decreased in the cerebellum in preconvulsive and convulsive phases. However, in the cerebral cortex there was a decrease in the activity of this enzyme only in the convulsive phase. The results suggest that glutamine acts more likely as a precursor for the neurotransmitter pool of glutamate, while ornithine serves more as a precursor for the neurotransmitter pool of GABA.     

Properties of Candida lipolytica mutants with the modified glyoxylate cycle and their ability to produce citric and isocitric acid

Summary Enzyme activities of the tricarboxylic acid (TCA) cycle and the anaplerotic pathways, as well as the cell cytology of two C. lipolytica mutants with the modified glyoxylate cycle and their parent strain were studied during the exponential growth phase on glucose or hexadecane.Among the TCA cycle enzymes, the key enzyme citrate synthase had the highest activity in all three strains grown on both substrates. NAD-dependent isocitrate dehydrogenase had the minimum activity. All strains had well-developed mitochondria.Pyruvate carboxylation was active in the wild strain and mutant 2 grown on glucose, where this reaction is the basic anaplerotic pathway for oxal-acetate synthesis; mutant 1 had actively functioning enzymes for both anaplerotic pathways — pyruvate carboxylase, isocitrate lyase and malate synthase.During hexadecane assimilation, the number of peroxisomes in all strains increased sharply, accompanied by a simultaneous increase in isocitrate lyase activity.The low activities of both isocitrate lyase and pyruvate carboxylase in mutant 2 give reason to believe that this strain has an additional pathway for oxalacetic acid synthesis during the assimilation of n-alkane.     

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.     

The effects of ammonium chloride and bicarbonate on the activity of glutaminase in isolated liver mitochondria.

1. Glutamine hydrolysis in liver mitochondria was studied by measuring the production of glutamate under conditions where this compound could not be further metabolized. 2. Glutaminase activity in intact mitochondria was very low in the absence of activators. 3. Glutamine hydrolysis was markedly stimulated by NH4Cl and also by HCO3- ions. 4. The stimulation by each of these compounds was much decreased if the mitochondria were uncoupled. 5. Maximum rates of glutamine hydrolysis required the addition of phosphate. A correlation was observed between the activity of glutaminase in the presence of NH4Cl plus HCO3- and the intramitochondrial content of ATP. 6. In disrupted mitochondria, NH4Cl stimulated glutaminase to a much smaller extent than in intact mitochondria. The NH4Cl stimulation in disrupted mitochondria was much increased by the addition of ATP. KHCO3 also stimulated glutaminase activity in disrupted mitochondria, and ATP increased the magnitude of this stimulation. 7. It was concluded that maximum rates of glutaminase activity in liver mitochondria require the presence of phosphate, ATP and either HCO3- or NH4+. A comparison of the results obtained on intact and broken mitochondria indicates that these effectors have a direct effect on the glutaminase enzyme system rather than an indirect effect mediated by changes in transmembrane ion gradients or in the concentrations of intramitochondrial metabolites.