A rapid radioactive assay for glutamine synthetase, glutaminase, asparagine synthetase, and asparaginase (original) (raw)
1970, Analytical Biochemistry
https://doi.org/10.1016/0003-2697(70)90069-2
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Abstract
AI
This study presents a rapid radioactive assay technique for evaluating the activities of glutamine synthetase, glutaminase, asparagine synthetase, and asparaginase using ion-exchange resins. The proposed method improves upon existing assays by offering greater sensitivity and ease of use, facilitating further research in enzymatic reactions and nitrogen metabolism.
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Glucosylglycine, Asparagine, and Glutamine Metabolism Inescherichia Coli
Journal of Bacteriology
N-Glucosylglycine, like glycine, stimulated the synthesis of 4-amino-5-imidazolecarboxamide riboside by sulfadiazine-inhibited cultures of Escherichia coli, but, unlike glycine, it did not contribute its elements to carboxamide synthesis (Rogers, King, and Cheldelin, 1960). Therefore, glucosylglycine could be a glycine metabolite, but not in the pathway to the purines. To see whether glucosylglycine belongs in a biosynthetic sequence proceeding from glycine and to ascertain the subsequent members, mutant cultures of E. coli were sought which were either unable to synthesize glucosylglycine or to metabolize it. This report deals with such a study and with the possible role of glucosylglycine in the biosynthesis of asparagine and glutamine peptides. MATERIALS AND METHODS Glucosylglycine was prepared as the ethyl ester by the method of Wolfrom, Schuetz, and Cavalieri (1949). Glucosylglycyl-L-asparagine was prepared as the methyl or ethyl ester by lyophilizing a solution containing equivalent amounts of glucose and the glycyl-L-asparagine ester.2 Glycyl-L-asparagine, DL-alanyl-DL-asparagine, and glycyl-DL-alanine were obtained from Mann Research Laboratories. Glycyl-L-glutamine was prepared from carbobenzoxyglycyl-L-glutamic acid (Mann) by the method of Thierfelder and von Cramm (1919), and methionine sulfoxide was prepared by the method of Lepp and Dunn (1955). Lycomarasmin3 was generously provided
Biochemistry, 1994
The role of Asp-50 and Glu-327 of Escherichia coli glutamine synthetase in catalysis and substrate binding has been interrogated by construction of site-directed mutants at these positions. Steadystate and rapid-quench kinetic methods were used to elucidate contributions of Asp-50 and Glu-327 to the K , values of all three substrates, ATP, glutamate, and NH4+, as well as to the enzymatic kat value. Kinetic constants were obtained for the D50A enzyme using both Mg2+ and Mn2+ as activating metal ions; the data reveal that Asp-50 has a significant role in both substrate binding and catalysis as reflected by the increases in the K , values for NH4+ and the destabilization of both the ground state and the transition state for phosphoryl transfer. The D50E mutant was found to have activity with Mn2+ but very low activity with Mg2+, the physiologically important metal ion. The ka,/Km values for all three substrates were substantially altered by changing Asp to Glu. The steady-state results for the E327A mutant indicate a decreased k,,lK, value for NH4+ compared to that of the wild-type enzyme. The E327A-Mg2+ enzyme destabilizes the ground state of the ternary complex (E-ATP-Glu-NHd+) and the transition state for phosphoryl transfer while the E327A-Mn2+-enzyme provides greater stabilization for the ATP and glutamate complexes but destabilizes phosphoryl transfer steps in the ternary complex. Overall, these results suggest that Asp-50 is likely involved in binding NH4+ and may also play a role in catalyzing deprotonation of NH4+ to form NH3. Glu-327 participates in lowering the free energy of the transition state involved in formation of the positively charged tetrahedral adduct resulting from the condensation of y-glutamyl phosphate and NH3.
Journal of Pharmaceutical Sciences, 2002
Our objective was to compare the relative rates of asparaginyl and glutaminyl deamidation in fragment 22-29 of the polypeptide hormone glucagon in acidic aqueous solutions. Reaction mixtures containing 22-29 (FVQWLMNT) or its degradation products were degraded at 608C in dilute hydrochloric acid or phosphate buffer in the pH range 1-3. Degradation products were separated by high-performance liquid chromatography and identified by amino acid sequencing, amino acid analysis, liquid chromatography-mass spectrometry (LC-MS), and matrix-assisted laser desorption and ionization (MALDI). Nine major degradation products were identified, including asparaginyl and glutaminyl deamidated forms, aspartyl peptide cleavage of the asparaginyl deamidated products, and a cyclic imide intermediate. The pH dependences of rate constants for individual pathways were consistent with acid catalysis. Previous investigators have reported a greater susceptibility of asparagine residues to deamidation in neutral and alkaline solutions due to the formation of a more stable five-membered succinimide intermediate. It has been suggested that asparagine may be more labile under acidic conditions also. We have observed a more facile deamidation for the glutamine residue under the acidic condition studied. It is proposed that the lower reactivity of the asparagine residue may be due to a decreased electrophilicity of its side chain carbonyl carbon imparted by a parallel cleavage pathway at this residue.
Fluorimetric assay of phosphate-activated glutaminase
Journal of Neuroscience Methods, 1983
A fluorimetric assay for the estimation of phosphate-activated glutaminase is presented. The liberated glutamate is separated from glutamine using a Dowex centrifugation technique allowing multiple samples to be rapidly analyzed. Glutamate is estimated fluorimetrically by reaction with o-phthaldialdehyde. Parameters for the assay were worked out based upon characterization of human frontal cortex glutaminase. High phosphate-activated glutaminase was found in cultured human skin fibroblasts and amniotic fluid cells and rat frontal cortex and striatum. Human caudate nucleus and frontal cortex activity was variable, but related in an exponential manner. Human and rat liver activity was markedly lower than brain activity.
The Journal of biological chemistry, 1973
The r,-glutamine analogs, L-albizziin (L-a-amino+ureidopropionic acid), S-carbamyl-L-cysteine, and O-carbamyl-Lserine were found to be substrates for purified rat liver glutamine transaminase, and the cr-keto acid product formed in each case was found to cyclize to a lactam analogous in structure to the cyclic form of ar-ketoglutaramic acid (Z-pyrrolidone-5-hydroxy-5-carboxylic acid). Evidence was obtained that the initial product of transamination of albizziin, S-carbamylcysteine, and O-carbamylserine are the corresponding cr-keto acids, which were found to be converted by w-amidase (followed by spontaneous decarboxylation) to &aminopyruvate, P-mercaptopyruvate, and P-hydroxypyruvate, respectively. Incubation of the glutamine analogs with L-amino acid oxidase from Crotalus adamanteus venom gave the corresponding cyclic lactam forms; the products obtained from albizziin and S-carbamyl-cysteine were rapidly and irreversibly dehydrated in acid or base to yield Z-imidazolinone-4-carboxylic acid and 2-thiazolinone-4-carboxylic acid, respectively. Neither a-ketoglutaramate nor Z-oxazolidone-4-hydroxy-4-carboxylic acid (which was isolated as the corresponding barium salt) was dehydrated under these conditions.
Determination of aspartate aminotransferase activity by high-performance liquid chromatography
Journal of chromatography. B, Biomedical applications, 1994
A sensitive and reproducible assay of aspartate aminotransferase activity based on UV detection of the reaction products after their separation by HPLC is described. The main advantage is the direct measurement of the enzyme activity as micromoles of product (glutamate) formed within a known period of time without any coupled reaction. Further, with the chromatographic method, all components of the reaction mixture are identified, allowing the reaction course to be controlled and the possible presence of side-reactions to be monitored.
Analytical Biochemistry, 1982
The amide nitrogen from L-glutamine has been isolated from an artificial plasma, in a form suitable for mass spectrometry, by a macromodification of the glutaminase reaction. The prior removal of free ammonia was carried out by alkaline aeration. When this was performed at 0°C for 3 h, spontaneous hydrolysis of glutamine was I .4%. Cross-contamination with nitrogen liberated from the amide group of asparagine can be avoided by preincubation with asparaginase for 2 h and removal of the freed ammonia prior to reacting with glutaminase. Hydrolysis of glutamine during this step is 12%. Measurements of enrichment can be made on samples yielding more than 1 rmol of glutamine amide-derived ammonia.
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Journal of Pharmacological Methods, 1978
Two ion-exchange micro-methods, sensitive to less than 4.5 pmoles of product formed per min, are described and evaluated for rapid and accurate measurement of the radioactively labeled CABA formed in assays of glutamic acid decarboxylase activity. Each of these provides, in comparison to extant assays: (a) a marked increase in sensitivity; (b) a marked decrease in the time required to process the samples; and (c) assurance the measured radioactivity is, in fact, in GABA. The first assay employs a small column of the anion-exchange resin AG-1 x 8-fluoride and provides quantitative recovery of GABA in the column effluent with blanks of 0.02% of the input counts of glutamate. The second method, which is used when glutamine is formed in the reactions, employs a column of cation-exchange resin to adsorb the GABA, which is subsequently eluted and counted directly. These methods avoid the problem of nonstoichiometry of 14C02 and GABA production which is present in assays in which only the evolution of COZ is measured, and they provide the opportunity to measure GABA formation in either the classical glutamate decarboxylase reaction or in the enzymatic conversion of an active contaminant which is sometimes present in the radioactive substrate. The data are consistent with the possibility that the active contaminant may be a better substrate than glutamate for the brain enzyme which forms GABA.
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Biochimica et Biophysica Acta (BBA) - General Subjects, 1985
The short-term metabolic fate of labeled nitrogen derived from [t3N]ammonia or from L-[amide-13Nlglutamine was determined in murine tumors known to be resistant (Ridgeway Osteogenic Sarcoma (ROS)) or sensitive (Sarcoma-180 (S-180)) to glutaminase therapy. At 5 rain after intraperitoneal injection of [ t3N]ammonia or of L-[am/de-13N]glutamine, only about 0.7% of the label recovered in both tumors was in protein and nucleic acid. After [13N]ammonia administration, most of the label (over 80%) was in a metabolized form; a large portion of this metabolized label (50-57%) was in the urea fraction with a smaller amount in glutamine (37-42%). The major short-term fate of label derived from L-[amide-t3N]glutamine was incorporation into components of the urea cycle with smaller amounts in the acidic metabolites and in acidic amino acids. No labeled urea was found during in vitro studies in which S-180 tumor slices were incubated with [t3N]ammonia, suggesting that the [t3N]urea formed in the tumor in the in vivo experiments was not due to de novo synthesis through carbamyi phosphate in the tumor. Both tumors exhibited very low glutamine synthetase activity. Following glutaminase treatment, glutamine synthetase and ~,-glutamyltransferase activities, while remaining low, increased in the resistant tumor but not in the sensitive tumor; this increase may be related to the insensitivity of the ROS tumor toward glutaminase treatment.
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Comparative studies of glutamine transaminases from rat tissues
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Inhibition of Glutamine Synthetase Triggers Apoptosis in Asparaginase-Resistant Cells
Cellular Physiology and Biochemistry, 2005
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Synthesis and Activation of Asparagine in Asparagine Auxotrophs of Saccharomyces cerevisiae
European Journal of Biochemistry, 1979
l-Asparagine synthesis in Saccharomyces cerevisiae is performed by a glutamine-dependent asparagine synthetase of the type found in higher organisms.Auxotrophy for asparagine has been obtained in two classes of mutants. In class I, asparagine synthetase activity is cancelled. These mutants combinetwo mutations, asnA− and asnB−. Neither asnA− nor asnB− mutation alone. leads to total auxotrophy. Partial auxotrophy as well as a strong decrease in enzyme activity result from asnA− mutation. No change is detectable in cells with the asnB− mutation alone. This, and Jones' report [J. Bacteriol. 134, 200–207 (1978)] of auxotrophy resulting from the combination of two mutations, are strong supports for asparagine synthesis being an unusual biosynthetic operation. In class II, auxotrophy results from a single mutation which leads to a modification of the efficiency of the asparaginyl-tRNA synthetase (asnRS− mutation). This auxotrophy is cancelled if asparaginase I activity (the only one present in ∑ 1278b wild type) is cancelled by casnI− mutation. This latter mutation allows an increase in the asparagine pool which is able to compensate for the asparaginyl-tRNA synthetase partial defect of the asnRS− mutant.
Journal of Neurochemistry, 2004
The source of nitrogen (N) for the de novo synthesis of brain glutamate, glutamine and GABA remains controversial. Because leucine is readily transported into the brain and the brain contains high activities of branched-chain aminotransferase (BCAT), we hypothesized that leucine is the predominant N-precursor for brain glutamate synthesis. Conscious and unstressed rats administered with [U-13 C] and/or [ 15 N]leucine as additions to the diet were killed at 0-9 h of continuous feeding. Plasma and brain leucine equilibrated rapidly and the brain leucine-N turnover was more than 100%/min. The isotopic dilution of [U-13 C]leucine (brain/ plasma ratio 0.61 ± 0.06) and [ 15 N]leucine (0.23 ± 0.06) differed markedly, suggesting that 15% of cerebral leucine-N turnover derived from proteolysis and 62% from leucine synthesis via reverse transamination. The rate of glutamate synthesis from leucine was 5 lmol/g/h and at least 50% of glutamate-N originally derived from leucine. The enrichment of [5-15 N]glutamine was higher than [ 15 N]ammonia in the brain, indicating glial ammonia generation from leucine via glutamate. The enrichment of [ 15 N]GABA, [ 15 N]aspartate, [ 15 N]glutamate greater than [2-15 N]glutamine suggests direct incorporation of leucine-N into both glial and neuronal glutamate. These findings provide a new insight for the role of leucine as N-carrier from the plasma pool and within the cerebral compartments.
l-Asparaginases from Citrobacter freundii
Biochimica et Biophysica Acta (BBA) - Enzymology, 1977
Three enzymes which catalyze the hydrolysis of L-asparagine have been identified in extracts of Citrobacter freundii. One of these (asparaginaseglutaminase (EC 3.5.1.1) also shows substantial glutaminase activity. This enzyme is extremely labile, is sensitive to inactivation by p-chloromercuribenzoate, and is not protected by dithiothreitol. A second enzyme (asparaginase B) is also sensitive to mercurials but is protected from inactivation by dithiothreitol. This enzyme has a relatively low affinity for L-asparagine (Kin = 1.7 " 10-3 M). The third enzyme (asparaginase A) is insensitive to inactivation by mercurials, is stable upon long term storage and has a relatively high affinity for L-asparagine (Km = 2.9 " 10-s M). This enzyme has been purified to homogeneity and has a molecular weight of approx. 140 000; the subunit weight being approx. 33 000. The C. freundii asparaginase A produced significant increases in the survival time of C3H/HE mice carrying the 6C3HED lymphoma tumor.
Journal of Neurochemistry, 1983
Intrastriatal injections of kainic acid (KA) were utilized to investigate the cellular localization of postsynaptic dopamine (DA) metabolism by type A and B monoamine oxidase (MAO) in rat striaturn. At 2 days postinjection, maximal degeneration of cholinergic and y-aminobutyric acid (GABA)ergic neurons was observed and found to be associated with a significant decrease in both type A and B M A 0 activity. However, over the next %day period, when only the process of gliosis appeared to be occurring, a selective return to control of type B MA0 activity was seen. When the metabolism of [3H]DA (10-1 M ) was examined in %day KA-lesioned rat striatal slices, an increase in [3H]dihydroxyphenylacetic acid (DOPAC) and [3H]homovanillic acid (HVA) formation was observed. The KA-induced elevation of [3H]DOPAC formation (but not [3H]HVA) was abolished by the DA neuronal uptake inhibitor nomifensine. This is consistent with earlier findings suggesting that HVA is formed exclusively within sites external to DA neurons, Experiments with clorgyline andlor deprenyl revealed that the relative roles of type A and B M A 0 in striatal DA d e a m ination remained unchanged following KA (9wo deamina_ tion by type A MAO) even though total deamination was substantially enhanced. At high concentrations of r 3 H ] D ~ ( 10-JM), deamination by type B MA0 could be increased to 30% of the total MA0 activity; however, this w a s observed in both control and KA-lesioned stnata. These results suggest that KA-sensitive neurons contain t y p e A andlor type B MAO. Moreover, whereas these neurons may metabolize DA, a major portion of postsynaptic DA deamination appears to occur within glial sites of rat striatal tissue. Furthermore, glial cells would appear to contain functionally important quantities of both t y p e A and B MAO. Key Words: Type A and B monoamine oxidase-Kainic acid-Dopamine metabolism. Schoepp D. D. a n d Azzaro A. J. Effects of intrastriatal kainic acid injection on [3H]dopamine metabolism in rat stnatal slices: Evidence for postsynaptic glial Cell metabolism by both the type A and B forms of monoamine oxidase. J .
Neuronal Glutamine Utilization: Pathways of Nitrogen Transfer tudied with [15N]Glutamine
Journal of Neurochemistry, 1989
Gas chromatography-mass spectrometry was used to evaluate the metabolism of [15N]glutamine in isolated rat brain synaptosomes. In the presence of 0.5 mM glutamine, synaptosomes accumulated this amino acid to a level of 25-35 nmol/mg protein at an initial rate >Y nmol/min/mg of protein. The metabolism of [ "N]glutamine generated "Nlabelled glutamate, aspartate, and y-aminobutyric acid (GABA). An efflux of both ['5N)glutamate and ["N]aspartate from synaptosomes to the medium was observed. Enrichment of I5N in alanine could not be detected because of a limited pool size. Elimination of glucose from the incubation medium substantially increased the rate and amount of [ "Nlaspartate formed. It is concluded that: (I) With 0.5 mM external glutamine, the glutaminase reaction, and not glutamine transport, determines the rate of metabolism of this amino acid. (2) The primary route of glutamine catabolism involves as-Glutamate, glutamine, and related amino acids are central components in brain metabolism and function (Hertz et al., 1983). Glutamate is the primary excitatory neurotransmitter in the CNS and glutamine appears to be an important source of glutamate released during depolarization (