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At least 19 recordsLinked to original sources

PubMed1975

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.

Alanine↗

PubMedDate not supplied

A comparative study of the renal glutaminase response to acidosis.

The rates of ammonia excretion and the activities of renal glutaminase were measured in the rat, dog, guinea pig and chicken. The rate of ammonia excretion and the activity of renal glutaminase were less in guinea pig than in the other species. Renal glutaminase from all four species was activated by phosphate, but the degree of activation varied between species. Many organic acids activated renal glutaminase in the rat, dog and chicken, but these compounds were without affect or decreased glutaminase activity in the guinea pig. The optimal pH for renal glutaminase was 7.4, 8.0 and 8.8 for the guinea pig, rat and dog respectively, while in the chicken a plateau was reached between pH 7.5 and 9.5. The activation of renal glutaminase by phosphate in the chicken was greater at sub-optimal pH values than in the plateau region. In the absence of an activator acidosis did not affect glutaminase activity in any species. When phosphate was present in the incubation medium acidosis increased glutaminase activity in the rat and chicken, but did not alter enzyme activity in the guinea pig or dog. Acidosis did not affect the optimal pH or the shape of the activity versus pH curve in any of the species.

Acidosis, Renal Tubular↗

PubMed1976

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.

Cold Temperature↗

PubMed1975

Enhancement of the glutaminase activity of carbamyl phosphate synthetase by alterations in the interaction between the heavy and light subunits.

Glutamine-dependent carbamyl phosphate synthetase (from Escherichia coli) was previously shown to be composed of a light subunit (molecular weight similar to 42,000) which has the binding site for glutamine and a heavy subunit (molecular weight similar to 130,000) which has binding sites for the other reactants and allosteric effectors. The subunits may be separated with retention of catalytic activities; only the separated light subunit exhibits glutaminase activity. The previous finding that storage of the native enzyme at pH 9 at 0 degrees increased its glutaminase activity by about 25-fold was further investigated; such storage markedly decreased the glutamine- and ammonia-dependent synthetase activities of the enzyme. Treatment of the enzyme with p-hydroxymercuribenzoate led to transient increase of glutaminase activity followed by inhibition. When the enzyme was treated with N-ethylmaleimide or with 5,5'-dithiobis-(2-nitrobenzoate), the glutaminase activity was increased by about 250-fold with concomitant loss of synthetase activities. The enhancement of glutaminase produced by storage of the enzyme at pH 9 was associated with intermolecular disulfide bond formation and aggregation of the enzyme. Aggregation also was observed after extensive treatment of the enzyme with 5,5'-dithiobis-(2-nitrobenzoate) or N-ethylmaleimide. However, a moderate increase of glutaminase activity (about 30-fold) was observed without aggregation under conditions in which one sulfhydryl group on the light subunit reacted with either reagent. The findings suggest that the increased glutaminase activities observed here are associated with structural changes in the enzyme in which the intersubunit relationship is altered so as to uncouple the catalytic functions of the enzyme and to facilitate access of water to the glutamine binding site on the light subunit.

Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzin↗

PubMed1975

Regulation of glutaminase levels in Escherichia coli.

Nitrogenous metabolites, cyclic adenosine 3':5'-monophosphate (cAMP), and the stage of culture growth all influence the levels of glutaminase A in Escherichia coli, but no variables in culture conditions alter the levels of glutaminase B. Growth of E. coli on culture media containing glucose and excess ammonia results in a rise in the level of glutaminase A as the cultures enter stationary phase; this rise is abolished by ammonia limitation. cAMP or glycerol reduce the level of glutaminase A. In mutants deficient in cAMP receptor protein, glutaminase A levels are unchanged by cAMP, but they are still susceptible to regulation by ammonia. We consider glutaminase B to be a constitutive enzyme, since its levels appear independent of nutritional conditions.

Aerobiosis↗

PubMed1976

Properties of rat kidney glutaminase enzymes and their role in renal ammoniagenesis.

Rat kidney contains two distinct glutaminase activities; the mitochondrial phosphate-dependent glutaminase and a second glutaminase activity associated with the brush border membrane which is maleate-activated and phosphate-independent. It has recently been shown that the phosphate-independent glutaminase is a partial reaction of gamma-glutamyl transpeptidase and that maleate activates this enzyme by blocking transpeptidation. The gamma-glutamyl transpeptidase in other rat tissues is also affected by maleate. This enzyme has at least a 100-fold greater affinity for glutathione or for glutathione derivatives than for glutamine, suggesting that under physiological conditions glutathione is the preferred substrate. With either type of substrate, maleate affects the Vmax of the reaction but not the Km. These findings suggest that this enzyme probably contributes very little to renal ammoniagenesis. In contrast, the phosphate-dependent glutaminase, whose activity increases 20 to 30-fold in the proximal convoluted tubule cells in response to metabolic acidosis, probably contributes significantly to renal ammoniagenesis. We have purified the rat kidney phosphate-dependent glutaminase and compared the phosphate activation and the phosphate-induced dimerization of the Tris form of this enzyme. There is an excellent correlation between increased activity and extent of dimerization as phosphate concentration is increased. The molecular weights of the Tris form are 1600000 and 316000 in the absence and presence of -1 M NaPO4, respectively. At saturating concentration of phosphate, increasing concentrations of chloride ion similarly reverse both activation and dimerization. These observations suggest that only the dimer form of the Tris enzyme is active.

Ammonia↗

PubMed1979

Inactivation of rat renal phosphate-dependent glutaminase with 6-diazo-5-oxo-L-norleucine. Evidence for interaction at the glutamine binding site.

Inactivation of rat renal phosphate-dependent glutaminase by 6-diazo-5-oxo-L-norleucine occurs only under conditions where the enzyme is catalytically active. The glutaminase activity and the rate of inactivation by the diazoketone exhibit very similar phosphate concentration-dependent activation profiles. Because of this phosphate dependency, it was not possible to differentiate an apparent protection by glutamine from the strong inhibition of inactivation caused by glutamate. The ability of glutamate to protect the glutaminase against inactivation is reversed by increasing concentrations of phosphate. The observed characteristics of inactivation by 6-diazo-5-oxo-L-norleucine differ considerably from those reported for the inactivation by L-2-amino-4-oxo-5-chloropentanoic acid. In addition, the presence of o-carbamoyl-L-serine was found to stimulate inactivation by 6-diazo-5-oxo-L-norleucine, but to protect the glutaminase against inactivation by the chloroketone. Preinactivation of the glutaminase by the diazoketone only slightly reduced the stoichiometry of binding of [5-14C]chloroketone. These observations suggest that 6-diazo-5-oxo-L-norleucine and L-2-amino-4-oxo-5-chloropentanoic acid interact with different sites on the glutaminase which are specific for binding glutamine and glutamate, respectively.

Animals↗

PubMed1975

[Effect of glutamic acid on the interrelationship of the effects of different activators of cerebral glutaminase].

When phosphate and tyroxine (activators of brain glutaminase) are used in small amounts, a potentiation of their stimulatory effect is observed. Higher concentrations exhibit an opposite effect. Glutamic acid has a strong inhibitory effect on all the activators of glutaminase given separately. The inhibitory effect of glutamate increases on lowering the pH. On the other hand the potentiation observed on adding two stimulators is increased greatly in the presence of glutamate. On the addition of tyroxine to other stimulators a greater potentiation and rise of glutaminase activity are observed. The potentiation, which occurs on the joint addition of phosphate and tyroxine, is raised with the increase of the amount of glutamic acid, while on the contrary on joining phosphate with other stimulators potentiation is reduced. Potentiation is variable and depends on the pH. Preincubation of brain mitochondrial fraction with guanidine chloride inhibits markedly the stimulatory effect of all the stimulators used, but their joint addition almost abolishes the potentiating effect. In the presence of glutamic acid, due to the increase of the cooperative effect between the two stimulators, glutaminase activity is greatly increased and sometimes its inhibitory effect is not even observed. The data obtained indicate that in brain glutamic acid in the presence of phosphate+thyroxine cannot be considered as an inhibitor of glutaminase and that the important factor here is not so much the absolute levels of the activators as their favorable combinations.

Animals↗

PubMed1975

Identity of maleate-stimulated glutaminase with gamma-glutamyl transpeptidase in rat kidney.

Gamma-Glutamyl transpeptidase was purified from rat kidney by a procedure involving Lubrol extraction, acetone precipitation, ammonium sulfate fractionation, treatment with bromelain, and column chromatography on DEAE-cellulose and Sephadex G-100. The final preparation (enzyme III), which exhibits a specific activity about 8-fold higher than that of the purified rat kidney transpeptidase previously obtained in this laboratory (enzyme I), was apparently homogeneous on polyacrylamide gel electrophoresis. Enzyme III is a glycoprotein containing 10% hexose, 7% aminohexose, and 1.5% sialic acid; a tentative molecular weight value of about 70,000 was obtained by gel filtration. Enzyme III has a much lower molecular weight and a different amino acid and carbohydrate content than the less active rat kidney transpeptidase preparation previously obtained, but obtained, but the catalytic properties of these preparations are virtually identical. It is suggested that bromelain treatment may liberate the transpeptidase from a brush border complex that contains other proteins. An improved method is described for the isolation of the higher molecular weight form of the enzyme (enzyme I) in which affinity chromatography on concanavalin A-Sephrose is employed. The purified transpeptidase (enzyme III) is similar to the phosphate-independent maleate-stimulated glutaminase preparation obtained from rat kidney by Katunuma and colleagues with respect to amino acid and carbohydrate content, apparent molecular weight, and relative transpeptidase and maleate-stimulated "glutaminase" activities. Both of these enzyme preparations are much more active in transpeptidation reactions with glutathione and related gamma-glutamyl compounds than with glutamine. In the absence of maleate, the enzyme catalyzes the utilization of glutamine (by conversion to gamma-glutamylglutamine, glutamate, and ammonia) at about 2% of the rate observed for catalysis of transpeptidation between glutathione and glycylglycine; the utilization of glutamine occurs about 8 times more rapidly in the presence of 0.1 M maleate. The transpeptidation and maleate-stimulated glutaminase reactions catalyzed by both enzyme preprations are inhibited by 5 mM L-serine in the presence of 5 mM sodium borate. Studies on gamma-glutamyl transpeptidase and maleate-stimulated glutaminase in the kidneys of fetal rats, newborn rats, and rats after weaning showed parallel development of these activities. The evidence reported here and earlier work in this laboratory strongly support the conclusion that maleate-stimulated glutaminase activity is a catalytic function of gamma-glutamyl transpeptidase. The studies on the ontogeny of gamma-glutamyl transpeptidase and other data are considered in relation to the proposal that this enzyme is involved in amino acid and peptide transport. Its possible role in renal formation of ammonia is also discussed.

Aging↗

PubMed1976

Purification and properties of a highly potent antitumor glutaminase-asparaginase from Pseudomonas 7Z.

Crystalline glutaminase-asparaginase which is effective against solid as well as ascites tumors was prepared from soil isolate organism Pseudomonas 7A. This enzyme has a ration of Vmax for L-glutamine and L-asparagine of 2.0. The presence of glutamic acid in the growth medium is essential for optimal enzyme production and glucose inhibits the production of glutaminase-asparaginase. The purification procedure provides an overall yield of 40 to 45% from crude cell extract to homogeneous glutaminase-asparaginase and is adaptable to large scale production of the enzyme. The specific activity of homogeneous enzyme is 160 +/- 15 i.u./mg of protein and the E1% 280 is 9.8. No disulfide or sulfhydryl groups appear to be present on the enzyme. The isoelectric point of glutaminase-asparaginase by isoelectric focusing on ampholine polyacrylamide gel plates is 5.8. The Km values for L-glutamine and L-asparagine are 4.6 and 4.4 X 10(-6) M, respectively. The enzyme catalyzes the hydrolysis of the D isomers of glutamine and asparagine at 87 and 69% the rate of the respective L isomers. L-Glutamic acid gamma-monohydroxamate is hydrolyzed at approximately the same rate as L-glutamine. The enzyme is not inhibited by ethylenediaminetetraacetate (0.1 mM), L-glutamate (30 mM), or L-aspartate (30 mM). Ammonium sulfate (10 mM) inhibits the enzymatic activity. The plasma half-life of Pseudomonas 7A glutaminase-asparaginase if 13 hours in normal mice and 43 hours in mice infected with the lactate dehydrogenase-elevating virus.

Amino Acids↗

PubMed1978

Kinetics of uptake and activity in mouse liver of glutaminase coupled to desialated orosomucoid.

Desialised orosomucoid (alpha-1-acidic glycoprotein) was coupled to Pseudomonas 7A glutaminase-asparaginase by glutaraldehyde, iodinated and injected into mice. The half-life of radioactivity and glutaminase activity in plasma was about 7 min. Radioactivity and glutaminase activity in the liver reached a peak at about 20 min. The radioactivity in liver then declined with a half-life of about 20 min. Enzyme activity in liver declined with a half-life of about 10 min. The ratio of enzyme activity to radioactivity was lower in the liver than in plasma at all times during the experiment, indicating rapid hepatic inactivation of the enzyme. Uptake into the liver could be blocked by excess desialised orosomucoid. Glutamine levels in the liver were about 10% of normal for 44 min but returned to 50% of normal by 93 min. Intestines, kidney and spleen failed to exhibit any appreciable uptake of desialated orosomucoid glutaminase-asparaginase.

Animals↗

PubMed1976

The mode of activation and regulation of glutaminase in intact kidney mitochondria.

Experiments were carried out with isolated pig renal-cortex and rat kidney mitochondria. Respiration, glutaminase activity and swelling of the mitochondria were followed by an oxygen electrode, an NH+4-sensitive electrode and spectrophotometrically. The inhibitors of the transport of glutamate (avenaciolide) and phosphate (mersalyl) across the inner mitochondrial membrane were employed. On the basis of the experimental findings we made the following conclusions: 1. There are two mechanisms by which glutamate can leave the inner space of kidney mitochondria: (a) an electrogenic efflus coupled to the respiration driven proton translocation and the presence of a membrane potential (positive outside), and (b) an electroneutral glutamate-hydroxyl exchange which is inhibited by avenaciolide and which operates in both directions. 2. The activation of glutaminase in intact mitochondria by phosphate occurs only if the activator moves across the inner mitochondrial membrane. The enzyme can be activated even when the movement of phosphate is inhibited provided that the mitochondria are energized. 3. Glutaminase is intimely associated with the inner mitochondrial membrane so that the part of the enzyme with the binding site for phosphate is embedded in the membrane, whereas the part with the binding sites for the substrate and glutamate is exposed toward the matrix space. The mechanism of allosteric activation of glutaminase and of the efflux of glutamate from mitochondria are probably the crucial factors which regulate the enzyme activity, and, consequently, renal ammoniagenesis in vivo.

Animals↗

PubMed1979

Cause of subunit heterogeneity in purified rat renal phosphate-dependent glutaminase.

When electrophoresed on polyacrylamide gels in the presence of sodium lauryl sulfate, highly purified rat renal phosphate-dependent glutaminase exhibits subunits which range in molecular weight from 57,000 to 75,000. Peptide mapping of the separated subunits following limited proteolysis in the presence of sodium lauryl sulfate shows that all of the various subunits are related in structure. The glutaminase, immunoprecipitated from Triton X-100-solubilized mitochondria, is composed primarily of subunits which have molecular weights of 83,000. In addition, the series of smaller subunits is generated during storage of the Triton-solubilized glutaminase at 4 degrees C. These results indicate that the heterogeneity of subunit size found in the purified glutaminase results from a noninactivating partial proteolysis of the native form of the enzyme.

Animals↗

PubMed1978

Covalent interaction of L-2-amino-4-oxo-5-chloropentanoic acid with rat renal phosphate-dependent glutaminase. Evidence for a specific glutamate binding site and of subunit heterogeneity.

Rat renal phosphate-dependent glutaminase is rapidly inactivated by incubating with L-2-amino-4-oxo-5-chloropentanoic scid. Concentrations of phosphate, which increase the glutaminase activity, decrease the rate of inactivation by chloroketone. In addition, inactivation is not blocked by glutamine. Instead, glutamate was shown to specifically reduce the rate of chloroketone inactivation. Upon sodium lauryl sulfate-polyacrylamide gel electrophoresis, the purified glutaminase preparation exhibits at least five protein staining bands which range in molecular weight from 57,000 to 75,000. Studies with 14C-labeled chloroketone indicate that this reagent reacts with each of these peptides. The mean stoichiometry of binding was calculated to be 1.3 mol/mol of enzyme. Therefore, these results indicate that the glutaminase may contain a specific site for binding glutamate and that the purified enzyme consists of a series of related peptides which may have resulted from partial proteolysis.

Amino Acids↗

PubMed1978

Amino acid sequence of the diazooxonorleucine binding site of Acinetobacter and Pseudomonas 7A glutaminase--asparaginase enzymes.

Acinetobactor glutaminase-asparaginase was treated with [6-14C]diazo-5-oxonorleucine, reduced with sodium borohydride, and cleaved with cyanogen bromide. Radioactivity was present only in a 96-residue-N-terminal peptide which eluted as the second peptide peak on Sephadex G-50. Radioactivity was released with the threonine in position 12 during automatic sequencing of this peptide. The amino acid sequence of a 60-residue tn-terminal segment and a 16-residue C-terminal segment of this peptide was determined. Pseudomonas 7 A glutaminase-asparaginase was treated with [6-14C]diazo-5-oxonorleucine and reduced with sodium borohydride. Radioactivity was released with the threonine in residue 20 during automatic sequencing of the whole enzyme. Analysis of 26 N-terminal residues showed that an 8-residue segment containing the radioactive threonine was identical with that in Acinetobacter glutaminase-asparaginase and in Escherichia coli asparaginase. Additional identical residues were noted in the N-terminal regions of these enzymes.

Acinetobacter↗

PubMed1975

Glutamine synthetase, glutaminase and phosphodiesterase activities in brain under hypoxia: in vitro effect of cortisol, GABA and serotonin on glutamine synthetase.

The effect of hypobaric hypoxia on the activities of glutamine synthetase, glutaminase and cyclic 3'5' AMP phosphodiesterase in rat brain was studied after exposure to 25,000' for 6 h. Glutamine synthetase activity was increased in all the regions of brain studied, and addition of gamma amino butyric acid, serotonin and cortisol in vitro produced a differential response. Glutaminase activity decreased in the whole brain. Cyclic 3'5' AMP phosphodiesterase activity decreased in cerebellum, medulla, hypothalamus and pituitary showing an accumulation of cyclic 3'5' AMP in these regions. The results suggest that glutamine synthesis and degradation are regulated in the central nervous system by cyclic AMP and cortisol: Gamma aminoburyric acid and other compounds can modulate the activity of glutamine synthetase and glutaminase.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1978

[Biological properties of an asparaginase-glutaminase preparation from Pseudomonas fluorescens in cell cultures].

Specific L-asparaginase activity and non-specific cytotoxicity of asparaginase-glutaminase preparation from Pseudomonas fluorescens were studied. Two cell lines, i.e. the asparaginase-dependent (Berkitt lymphoma cells) and the asparaginase-independent (the ovary cancer cells) were used as the test-system. Incorporation of 3H-timidine into DNA was used as the criterion of the drug effect on the cells. Krasnitin was used as the reference preparation. The preparation of asparaginase-glutaminase was inferior to krasnitine by its specific antitumour asparaginase activity and superior to it by the general cytotoxicity in the cells of CaOv. With the help of the above test-system it is possible to study the specific asparaginase activity of the drugs containing L-asparaginase. For studying the specific glutaminase properties it is necessary to develop another cell test-system.

Antibiotics, Antineoplastic↗

PubMed1979

Treatment of L5178Y tumor-bearing BDF1 mice with a nonimmunogenic L-glutaminase-L-asparaginase.

An L-glutaminase-L-asparaginase from Achromobacter has been rendered nonimmunogenic by the covalent attachment of polyethylene glycol (PEG) to nonessential amine groups of the enzyme. PEG-L-glutaminase-L-asparaginase exhibits a greatly enhanced half-life in the bloodstream compared to the unmodified enzyme in normal mice, and is effective in prolonging the survival of BDF1 mice inoculated ip with L5178Y cells. PEG-L-glutaminase-L-asparaginase appears rapidly in the blood following ip injection.

Amidohydrolases↗