MODULATORS OF GLUTAMINASE ACTIVITY

Abstract— The activating effect of GTP on particulate preparations of glutaminase from rat brain or rat kidney was competitively inhibited by cyclic guanosine 3′,5′-monophosphate (_c-GMP). Similarly, the effect of ATP was inhibited by cyclic adenosine 3′,5′-monophosphate (_c-AMP). In the absence of an added activator, the glutaminase activity of brain or kidney particles, if measurable, was also inhibited by _c-GMP and _c-AMP. GTP was a stronger activator than ATP, and _c-GMP was a stronger inhibitor than _c-AMP, for the enzyme from either tissue. The K₁, of _c-GMP was about 40 mM, that of _c-AMP was about 60 mM, as determined with a brain preparation. Soluble preparations of glutaminase from pig brain and pig kidney were activated, in decreasing order of efficiency, by riboflavin phosphate, GTP, ATP and orthophosphate. The relative potencies were similar for the soluble enzymes from brain and kidney and also in the particulate and soluble forms of the brain enzyme. The apparent K_m, values for the soluble brain enzyme were about the same (9–10 mM) whether riboflavin phosphate, GTP, ATP or 100 mM orthophosphate were used as activators. The Km increased with concentrations of orthophosphate lower than 100 mM, but was not significantly affected by changes in the concentrations of the other activators. Results for the kidney enzyme were similar, but the K_m, tended to be somewhat lower. The estimation of the apparent K_A with either the brain or kidney enzyme preparation indicated that affinities and activating efficiency were related. The activating effects of carboxylic acids on the soluble brain enzyme were similar to those on the particulate brain enzyme in terms of some correlation to the number of carboxylic groups per molecule and the potentiation by orthophosphate, but in the soluble kidney enzyme these effects were less marked or absent.     

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.     

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.     

The effect of acetyl-coenzyme A on phosphate-activated glutaminase from pig kidney and brain

Phosphate-activated glutaminase (EC 3.5.1.2; l-glutamine amidohydrolase) purified from pig kidney and brain is activated by CoA and short-chain acyl-CoA derivatives. Acetyl-CoA is the most powerful activator (K(A) about 0.2mm). Acetyl-CoA is maximally effective in the absence of other activating anions such as phosphate and citrate, and at low glutamine concentrations. The negative co-operative substrate activation observed at pH7 becomes more pronounced in the presence of acetyl-CoA. Similarly to phosphate, acetyl-CoA produces at high protein concentrations a different type of activation, which is time-dependent, depends on protein concentration and is accompanied by an increase in the sedimentation coefficient. Acetyl-CoA, phosphate and citrate appear to have binding sites in common. No significant difference was observed between kidney and brain phosphate-activated glutaminase.     

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.     

Effect of lysine to alanine mutations on the phosphate activation and BPTES inhibition of glutaminase

The GLS1 gene encodes a mitochondrial glutaminase that is highly expressed in brain, kidney, small intestine and many transformed cells. Recent studies have identified multiple lysine residues in glutaminase that are sites of N-acetylation. Interestingly, these sites are located within either a loop segment that regulates access of glutamine to the active site or the dimer:dimer interface that participates in the phosphate-dependent oligomerization and activation of the enzyme. These two segments also contain the binding sites for bis-2[5-phenylacetamido-1,2,4-thiadiazol-2-yl]ethylsulfide (BPTES), a highly specific and potent uncompetitive inhibitor of this glutaminase. BPTES is also the lead compound for development of novel cancer chemotherapeutic agents. To provide a preliminary assessment of the potential effects of N-acetylation, the corresponding lysine to alanine mutations were constructed in the hGAC_Δ1 plasmid. The wild type and mutated proteins were purified by Ni⁺-affinity chromatography and their phosphate activation and BPTES inhibition profiles were analyzed. Two of the alanine substitutions in the loop segment (K₃₁₁A and K₃₂₈A) and the one in the dimer:dimer interface (K₃₉₆A) form enzymes that require greater concentrations of phosphate to produce half-maximal activation and exhibit greater sensitivity to BPTES inhibition. By contrast, the K₃₂₀A mutation results in a glutaminase that exhibits near maximal activity in the absence of phosphate and is not inhibited by BPTES. Thus, lysine N-acetylation may contribute to the acute regulation of glutaminase activity in various tissues and alter the efficacy of BPTES-type inhibitors.     

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.     

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

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

PIG BRAIN GLUTAMINASE: PURIFICATION AND IDENTIFICATION OF DIFFERENT ENZYME FORMS

—Pig brain glutaminase (EC 3.5.1.2 L-glutamine amidohydrolase) has been purified about 5000-fold from acetone powder. Glutaminase exists in different molecular forms, dependent on the ionic composition of the buffer. The three main forms are similar to those of kidney glutaminase and therefore called the tris-HCl enzyme, the phosphate enzyme, and the phosphate-borate enzyme. The sedimentation coefficients, as estimated by sucrose gradient technique, are 7·3, 8·7, and 53, respectively. Glutaminase has a pH optimum of about 9, but the pH curves of the tris-HCl enzyme and the phosphate-borate enzyme have different shapes. The apparent pK₁ of the tris-HCl enzyme-substrate complex is similar to pK₂ of inorganic phosphate, the apparent pK₂ of both the tris-HCl and the phosphateborate enzyme complexes is similar to pK₂ of glutamine. By use of the electron microscope we were able to see the phosphate-borate enzyme.     

Effects of neuroleptics on glutaminase from rat synaptosomes

Phosphate-activated glutaminase was isolated from synaptosomes from three areas of rat brain. Glutamine utilization phosphate activation and inhibition by glutamate or ammonia were assessed in the absence or presence of haloperidol, chlorpromazine, or clozapine. All three drugs (at 1 micromolar concentration) elevated theK _m for glutamine using preparations from the amygdala, hippocampus, or striatum. They interfered with phosphate activation only in the amygdala preparation. No drug affected end-product inhibition. The data suggest that neuroleptics may depress the release of glutamic acid from synaptosomes by interfering with the activation of glutaminase by phosphate.     

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)     

Stimulation of mammalian adenylate cyclase activity with guanosine 5′-tetraphosphate and other guanine nucleotides

Guanosine 5′-tetraphosphate (GTP₄) stimulated mammalian adenylate cyclase activity at concentrations down to 1 μM. Greater stimulatory activity was apparent with lung than with heart, brain or liver from the rat. At a concentration of 0.1 mM, GTP₄ stimulated lung adenylate cyclase activity from rat, guinea pig and mouse about four-fold. Other guanine nucleotides such as GTP, GDP, GMP, guanosine 3′, 5′-monophosphate and 5′-guanylylimidodiphosphate (GMP · PNP) also stimulated mammalian adenylate cyclase activity. GMP · PNP irreversibly activated, whereas GTP₄ and GTP reversibly activated adenylate cyclase. Adenosine 5′-tetraphosphate (ATP₄) stimulated rat lung and liver but inhibited rat heart and brain adenylate cyclase activities. Lung from guinea pig and mouse were not affected by ATP₄. The formation of cyclic AMP by GTP₄-stimulated rat lung adenylate cyclase was verified by Dowex-50 (H⁺), Dowex 1-formate and polyethyleneimine cellulose column chromatography. GTP₄ was at least three times more potent than 1-isoproterenol in stimulating rat lung adenylate cyclase activity. The β-adrenergic receptor antagonist propranolol blocked the effect of 1-isoproterenol but not that of GTP₄, thus, suggesting that GTP₄ and β-adrenergic agonists interact with different receptor sites on membrane-bound adenylate cyclase. Stimulation of rat lung and liver adenylate cyclase activities with 1-isoproterenol was potentiated by either GTP₄ or GMP. PNP, thus indicating that GTP₄ resembles other guanine nucleotides in their capacity to increase the sensitivity of adenylate cyclase to β-adrenergic agonists. Stimulation of adenylate cyclase activity by guanine derivatives requires one or more free phosphate moieties on the 5 position of ribose, as no effect was elicited with guanine, guanosine, guanosine 2′-monophosphate, guanosine 3′-monophosphate or guanosine 2′,5′-monophosphate. Ribose, ribose 5-phosphate, phosphate and pyrophosphate were inactive. Pyrimidine nucleoside mono-, di-, tri- and tetraphosphates elicited negligible effects on mammalian adenylate cyclase activity.     

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.     

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.     

Syncoilin is an intermediate filament protein in activated hepatic stellate cells

Hepatic stellate cells (HSCs) play an important role in several (patho)physiologic conditions in the liver. In response to chronic injury, HSCs are activated and change from quiescent to myofibroblast-like cells with contractile properties. This shift in phenotype is accompanied by a change in expression of intermediate filament (IF) proteins. HSCs express a broad, but variable spectrum of IF proteins. In muscle, syncoilin was identified as an alpha-dystrobrevin binding protein with sequence homology to IF proteins. We investigated the expression of syncoilin in mouse and human HSCs. Syncoilin expression in isolated and cultured HSCs was studied by qPCR, Western blotting, and fluorescence immunocytochemistry. Syncoilin expression was also evaluated in other primary liver cell types and in in vivo-activated HSCs as well as total liver samples from fibrotic mice and cirrhotic patients. Syncoilin mRNA was present in human and mouse HSCs and was highly expressed in in vitro- and in vivo-activated HSCs. Syncoilin protein was strongly upregulated during in vitro activation of HSCs and undetectable in hepatocytes and liver sinusoidal endothelial cells. Syncoilin mRNA levels were elevated in both CCl₄- and common bile duct ligation-treated mice. Syncoilin immunocytochemistry revealed filamentous staining in activated mouse HSCs that partially colocalized with α-smooth muscle actin, β-actin, desmin, and α-tubulin. We show that in the liver, syncoilin is predominantly expressed by activated HSCs and displays very low-expression levels in other liver cell types, making it a good marker of activated HSCs. During in vitro activation of mouse HSCs, syncoilin is able to form filamentous structures or at least to closely interact with existing cellular filaments.     

TIME AND TEMPERATURE DEPENDENT ACTIVATION OF PIG BRAIN GLUTAMINASE

Abstract— A time-dependent activation of the tris-HC1 enzyme form of pig brain glutaminase (EC 3.5.1.2, L-glutamine amidohydrolase) by phosphate, phosphate-borate and carboxylic acids is described. This time-dependent activation increases with increased protein concentration and also with temperature. The sedimentation behaviour of the various activated enzyme preparations is described. The Arrhenius plot for the triq-HC1 enzyme is curved, indicating a reversible equilibrium between two enzyme forms, whereas the plot obtained for the phosphate-borate enzyme was a straight line. The apparent activationenergy has been determined.