Rat liver glutaminase. Regulation by reversible interaction with the mitochondrial membrane

Partially purified rat liver mitochondrial glutaminase shows a sigmoidal dependence on glutamine concentration, and an absolute requirement for inorganic phosphate as activator. Reconstitution with a mitochondrial membrane fraction changes the kinetic properties of the enzyme making the glutamine dependence more hyperbolic and reducing the concentration of phosphate required for half-maximum activation. Glutaminase activity in isolated mitochondria is known to be increased as a result of mitochondrial swelling. In mitochondria suspended in isotonic medium, the properties of glutaminase resemble of the isolated enzyme while in swollen mitochondria the kinetic properties revert to those exhibited by the enzyme in association with the mitochondrial membrane. It is postulated that mitochondrial glutaminase is regulated in situ by reversible association with the inner mitochondrial membrane which is mediated by mitochondrial swelling. This mechanism may explain the short-term hormonally induced activation of the enzyme observed in isolated hepatocytes.     

Rat hepatic glutaminase: purification and immunochemical characterization

A method for the purification of phosphate-activated glutaminase from the liver of streptozotocin-diabetic rats is described. The procedure involves solubilization of glutaminase activity from isolated mitochondria by sonication, followed by ammonium sulfate precipitation, polyethylene glycol precipitation, and sequential chromatography on DEAE, hydroxylapatite, and zinc-chelated resins. The enzyme was purified 600-fold to a specific activity of 31-57 U/mg protein. The purified enzyme has an apparent subunit molecular mass of 58,000-Da and is greater than 80% pure by scanning densitometry of sodium dodecyl sulfate-polyacrylamide gels. The purified enzyme has an apparent Km for glutamine of 17 mM and a pH optimum between 7.8 and 8.2. The physical and kinetic properties of this enzyme are similar to those of the enzyme from normal rat liver. Polyclonal antibodies raised against the enzyme specifically inhibit hepatic glutaminase activity and react primarily with a 58,000-Da peptide in liver fractions on immunoblots. These antibodies were used in equivalence point titrations and immunoblots to provide evidence for increased concentration of glutaminase protein in the liver of diabetic rats with no change in specific activity of the enzyme. In addition, the antibodies cross-react, at low affinity, with kidney-type glutaminases. On immunoblots, the antibodies did not react with fetal liver, mammary gland, or lung. Antibodies to rat hepatic glutaminase should prove useful as tools to study the long-term regulation of the enzyme.     

Digestion by serine proteases enhances salt tolerance of glutaminase in the marine bacterium <Emphasis Type="Italic">Micrococcus luteus</Emphasis> K-3

Salt-tolerant glutaminase (Micrococcus glutaminase, with an apparent molecular mass of 48.3 kDa, intact glutaminase) from the marine bacterium Micrococcus luteus K-3 was digested using protease derived from M. luteus K-3. The digestion products were a large fragment (apparent molecular mass of 38.5 kDa, the glutaminase fragment) and small fragments (apparent molecular mass of 8 kDa). The digestion was inhibited by phenylmethanesulfonyl fluoride (PMSF). Digestion of intact glutaminase by serine proteases including trypsin, elastase, lysyl endopeptidase, and arginylendopeptidase also produced the glutaminase fragment. The N-terminus of the glutaminase fragment was the same as that of intact glutaminase. The N-termini of two small fragments were Ala394 and Ala396, respectively. The enzymological and kinetic properties of the glutaminase fragment were almost the same as those of intact glutaminase except for salt-tolerant behavior. The glutaminase fragment was a higher salt-tolerant enzyme than the intact glutaminase, suggesting that Micrococcus glutaminase is digested in the C-terminal region by serine protease from M. luteus K-3 to confer salt tolerance on glutaminase.     

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.     

Engineering the substrate specificity of Escherichia coli asparaginase. II. Selective reduction of glutaminase activity by amino acid replacements at position 248

The use of Escherichia coli asparaginase II as a drug for the treatment of acute lymphoblastic leukemia is complicated by the significant glutaminase side activity of the enzyme. To develop enzyme forms with reduced glutaminase activity, a number of variants with amino acid replacements in the vicinity of the substrate binding site were constructed and assayed for their kinetic and stability properties. We found that replacements of Asp248 affected glutamine turnover much more strongly than asparagine hydrolysis. In the wild-type enzyme, N248 modulates substrate binding to a neighboring subunit by hydrogen bonding to side chains that directly interact with the substrate. In variant N248A, the loss of transition state stabilization caused by the mutation was 15 kJ mol(-1) for L-glutamine compared to 4 kJ mol(-1) for L-aspartic beta-hydroxamate and 7 kJ mol(-1) for L-asparagine. Smaller differences were seen with other N248 variants. Modeling studies suggested that the selective reduction of glutaminase activity is the result of small conformational changes that affect active-site residues and catalytically relevant water molecules.     

Effects of Phospholipases on the Kinetic Properties of Rat Striatal Membrane-Bound Tyrosine Hydroxylase

Abstract: Rat striatal tyrosine hydroxylase can be isolated in both a soluble and a synaptic membrane-bound form. The membrane-bound enzyme, which exhibits lower K _ms for both tyrosine (7 μ M ) and reduced pterin cofactor (110 μ M ) relative to the soluble enzyme (47 μ M and 940 μ M , respectively), can be released from the membrane fraction with mild detergent, and concomitantly its kinetic properties revert to those of the soluble enzyme. Treatment of membrane-bound tyrosine hydroxylase with C. perfringens phospholipase C increased the K _m of the enzyme for tyrosine to 27 μ M and the V _max by 60% without changing the K _m for cofactor. In contrast, treatment of membrane-bound tyrosine hydroxylase with V. russelli phospholipase A₂ increased the K _m for tyrosine to 48 μ M increased the V _max and increased the K _m for cofactor to 560 μ M . The enzyme remained bound to the membrane fraction following both phospholipase treatments. Addition of phospholipids to treated enzyme could partially reverse the effects of phospholipase A₂ treatment, but not the effects of phospholipase C treatment. The kinetic properties of phospholipase-treated, detergent-solubilized tyrosine hydroxylase were identical to those of the control solubilized enzyme. Tyrosine hydroxylase appears to interact with synaptic membrane components to produce at least two separately determined consequences for the kinetic properties of the enzyme.     

Kinetic model of imidazologlycerol-phosphate synthetase from <Emphasis Type="Italic">Escherichia coli</Emphasis>

Based on the available experimental data, we developed a kinetic model of the catalytic cycle of imidazologlycerol-phosphate synthetase from Escherichia coli accounting for the synthetase and glutaminase activities of the enzyme. The rate equations describing synthetase and glutaminase activities of imidazologlycerol-phosphate synthetase were derived from this catalytic cycle. Using the literature data, we evaluated all kinetic parameters of the rate equations characterizing individually synthetase and glutaminase activities as well as the contribution of each activity depending on concentration of the substrates, products, and effectors. As shown, in the presence of 5 -phosphoribosylformimino-5-aminoimidazolo-4-carboxamideribonucleotide (ProFAR) and imidazologlycerol phosphate (IGP) glutaminase activity dominates over synthetase activity at sufficiently low concentrations of 5 -phosphoribulosylformimino-5-aminoimidazolo-4-carboxamideribonucleotide (PRFAR). Increased PRFAR concentrations resulted in decreased contribution of glutaminase activity and, consequently, increased the contribution of synthetase activity in the enzyme functioning.     

PRODUCTION AND OPTIMIZATION OF L-GLUTAMINASE ENZYME FROM Hypocrea jecorina PURE CULTURE

L-Glutaminase (L-glutamine amidohydrolase, EC 3.5.1.2) is the important enzyme that catalyzes the deamination of L-glutamine to L-glutamic acid and ammonium ions. Recently, L-glutaminase has received much attention with respect to its therapeutic and industrial applications. It acts as a potent antileukemic agent and shows flavor-enhancing capacity in the production of fermented foods. Glutaminase activity is widely distributed in plants, animal tissues, and microorganisms, including bacteria, yeasts, and fungi. This study presents microbial production of glutaminase enzyme from Hypocrea jecorina pure culture and determination of optimum conditions and calculation of kinetic parameters of the produced enzyme. The optimum values were determined by using sa Nesslerization reaction for our produced glutaminase enzyme. The optimum pH value was determined as 8.0 and optimum temperature as 50°C for the glutaminase enzyme. The Km and Vmax values, the kinetic parameters, of enzyme produced from Hypocrea jecorina, pure culture were determined as 0.491 mM for Km and 13.86 U/L for Vmax by plotted Lineweaver–Burk graphing, respectively. The glutaminase enzyme from H. jecorina microorganism has very high thermal and storage stability.     

Microbial glutaminase: biochemistry, molecular approaches and applications in the food industry

Glutaminase is widely distributed in microorganisms including bacteria, yeast and fungi. The enzyme mainly catalyzes the hydrolysis of γ-amido bond of -glutamine. In addition, some enzymes also catalyze γ-glutamyl transfer reaction. A highly savory amino acid, -glutamic acid and a taste-enhancing amino acid of infused green tea, theanine can be synthesized by employing hydrolytic or transfer reaction catalyzed by glutaminase. Therefore, glutaminase is one of the most important flavor-enhancing enzymes in food industries. In this review, subsequent to a discussion on the definition of glutaminase, the enzymatic properties, applications of glutaminase in the food industry, and occurrence and distribution of the enzyme are described. We then illustrate the gene cloning, primary structure, and 3D-structure of glutaminase. Finally, to facilitate the future applications of glutaminase in food fermentations, the mechanisms of action of salt-tolerant glutaminase are briefly discussed.     

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

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

Phosphate-independent glutaminase from rat kidney. Partial purification and identity with gamma-glutamyltranspeptidase.

Phosphate-independent glutaminase can be quantitatively solubilized from a microsomal preparation of rat kidney by treatment with papain. Subsequent gel filtration and chromatography on quaternary aminoethyl (QAE)-Sephadex and hydroxylapatite yield a 200-fold purified preparation of this glutaminase. The purified enzyme also hydrolyzes gamma-glutamylhydroxamate and exhibits substrate inhibition at high concentrations of either glutamine or gamma-glutamyhydroxamate, which is partially relieved by increasing concentrations of maleate. Rat kidney phosphate-independent glutaminase reaction is catalyzed by the same enzyme which catalyzes the gamma-glutamyltranspeptidase reaction. The ratio of glutaminase to transpeptidase activities remained constant throughout a 200-fold purification of this enzyme. The observation that the phosphate0independent glutaminase and gamma-glutamyltranspeptidase activities exhibit coincident mobilities during electrophoresis, both before and after extensive treatment with neuraminidase, strongly suggests that both reactions are catalyzed by the same enzyme. This conclusion is strengthened by the observation that maleate and various amino acids have reciprocal effects on the two activities. Maleate increases glutaminase activity and blocks transpeptidation, whereas amino acids activate the transpeptidase but inhibit glutaminase activity. In contrast, the addition of both maleate and alanine resulted in a strong inhibition of both activities. Both activities exhibit a similar distribution in the various regions of the kidney. Recovery of maximal activities in the outer stripe region of the medulla is consistent with previous quantitative microanalysis which indicated that this glutaminase activity is localized primarily in the proximal straight tubule cells. The glutaminase and transpeptidase activities have different pH optima. Examination of the product specificity suggests that decreasing pH also promotes glutaminase activity and that below pH 6.0, this enzyme functions strictly as a glutaminase. Because of the localization of this activity on the brush border membrane, these resuts are consistent with the possibility that the physiological conditions induced by metabolic acidosis could convert this enzyme from a broad specificity transpeptidase to a glutaminase. Therefore, this enzyme could contribute to the increased renal synthesis of ammonia from glutamine which is observed during metabolic acidosis.     

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.     

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.     

Phosphate-dependent glutaminase in the human term placental mitochondria

The properties of placental glutaminase described in this paper, namely relatively high substrate affinity. Hill coefficient about 1.6, inhibition by glutamate, and the lack of activation by bicarbonate make the placental enzyme very similar to the "kidney type" glutaminase isoenzyme of rat tissues.     

Expression of functional human glutaminase in baculovirus system: affinity purification, kinetic and molecular characterization

Glutaminase catalyzes the hydrolysis of glutamine yielding stoichiometric amounts of glutamate plus ammonium ions. In mammals, there are two different genes encoding for glutaminase, known as liver (L) and kidney (K) types. The human L-type isoform expressed in baculovirus yielded functional recombinant enzyme in Sf9 insect cells. A novel affinity chromatography method, based on its specific interaction with a PDZ protein, was developed for purification. Kinetic constants were determined for the purified human isozyme, which showed an allosteric behaviour for glutamine, with a Hill index of 2.7 and S(0.5) values of 32 and 64 mM for high and low P(i) concentrations, respectively. Whereas the protein showed a low P(i) dependence typical for L-type glutaminases, the enzyme was unexpectedly inhibited by glutamate, a kinetic characteristic exclusive of K-type isozymes, and was slightly activated by ammonia, unlike the classical liver enzymes which show an absolute dependence on ammonia. Subcellular fractionation demonstrates that recombinant human glutaminase was targeted to both mitochondria and nucleus, and in both locations the protein was catalytically active. This is the first report of the expression of a functional L-type mammalian glutaminase enzyme. The study also provides a simple and efficient method for affinity purification of the recombinant enzyme. Moreover, the data imply that this human enzyme may represent a new isoform different from classical kidney and liver isozymes.     

Identification and properties of 2 forms of plant phospholipase D]

Effect of different factors (pH, temperature etc.) on the activity of phospholipase D is studied. It is found that plant phospholipase D has two forms: thermolabile (D1) and thermostable (Ds). Both forms are shown to differ in some characteristics (pH optimum, temperature optimum, calcium ions activation, activator effect, kinetic parameters, thermostavility). The contradictive literature data on phospholipase D properties are suggested to be due to the existence of two enzyme forms.     

Folate synthesis in plants: purification, kinetic properties, and inhibition of aminodeoxychorismate synthase

pABA (p-aminobenzoate) is a precursor of folates and, besides esterification to glucose, has no other known metabolic fate in plants. It is synthesized in two steps from chorismate and glutamine, the first step being their conversion into glutamate and ADC (4-aminodeoxychorismate). In Escherichia coli, two proteins forming a heterodimeric complex are required for this reaction, but, in plants and lower eukaryotes, a single protein is involved. The Arabidopsis enzyme was expressed in E. coli and was purified to homogeneity. The monomeric enzyme (95 kDa) catalyses two reactions: release of NH3 from glutamine (glutaminase activity) and substitution of NH3 for the hydroxy group at position 4 of chorismate (ADC synthase activity). The kinetic parameters of the plant enzyme are broadly similar to those of the bacterial complex, with K(m) values for glutamine and chorismate of 600 and 1.5 microM respectively. As with the bacterial enzyme, externally added NH3 was a very poor substrate for the plant enzyme, suggesting that NH3 released from glutamine is preferentially channelled to chorismate. The glutaminase activity could operate alone, but the presence of chorismate increased the efficiency of the reaction 10-fold, showing the interdependency of the two domains. The plant enzyme was inhibited by dihydrofolate and its analogue methotrexate, a feature never reported for the prokaryotic system. These molecules were inhibitors of the glutaminase reaction, competitive with respect to glutamine (K(i) values of 10 and 1 microM for dihydrofolate and methotrexate respectively). These findings support the view that the monomeric ADC synthase is a potential target for antifolate drugs.     

Physiological role of glutaminase activity in Saccharomyces cerevisiae

The participation of glutaminase activity in glutamine degradation was studied in a wild-type strain (S288C) of Saccharomyces cerevisiae. Evidence is presented that this strain has two glutaminase activities, a readily extractable form (glutaminase B) and a membrane-bound enzyme (glutaminase A). Glutaminase A and B activities could also be distinguished by their thermostability, pyruvate sensitivity and pH optimum. Glutaminase B activity was negatively modulated by some 2-oxo acids, and in vivo pyruvate accumulation inhibited this activity. A mutant strain (CN10) with an altered glutaminase B activity was isolated and partially characterized. Its glutaminase B activity was more sensitive to inhibition by pyruvate and 2-oxoglutarate than the wild type, thus resulting in inactivation of this enzyme in vivo. The physiological role of glutaminase activity is discussed with regard to the phenotype shown by the mutant strain.     

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