A rapid radioactive assay for glutamine synthetase, glutaminase, asparagine synthetase, and asparaginase (original) (raw)
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The Reaction of beta-Chloroglutamic Acid with Glutamate-Aspartate Transaminase
European Journal of Biochemistry, 1968
Porcine glutamate-aspartate transaminase catalyzes a @-elimination reaction with both the threo-and erythro-isomers of 8-chloroglutamate ; chloride, ammonia, and a-ketoglutarate are formed in equimolar amounts. The latter product was characterized as the 2,4-dinitrophenylhydrazone and by catalytic hydrogenation of this derivative to glutamic acid.
Anticancer research
Glutamine (Gln) deamination by asparaginase (ASNase) appears to contribute in the decrease of serum asparagine (Asn) levels and enhance leukemic cell apoptosis. The pharmacodynamic (PD) rationale is based on the role of Gln as the main amino group donor for Asn synthesis from aspartate by the enzyme asparagine synthetase (AS). Relationships between ASNase enzymatic activity and Asn or Gln levels were examined in 274 pairs of pre- and post-ASNase serum specimens from 200 high-risk acute lymphoblastic leukemia (ALL) patients from the Children's Cancer Group (CCG-1961). Data were analyzed according to a novel PD model based on previous best-fit projections (NONMEM) from the CCG-1962 standard-risk ALL study. The PD results from high-risk and standard-risk ALL patients were superimposable. The percentages of Asn and Gln deamination were predicted by ASNase activity in patients' sera. Pharmacodynamic analyses strongly suggested that > 90% deamination of Gln must occur before op...
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.
Purification and characterization of γ-glutamylcysteine synthetase from Ascaris suum
Molecular and Biochemical Parasitology, 1995
T-Glutamyltransferase ((5-glutamyl)-peptide:amino-acid 5-glutamyltransferase, EC 2.3.2.2.) from rat pancreas has been purified to homogeneity and shown to be a glycoprotein of apparent molecular weight 68000, composed of one heavy and one light subunit, with respective molecular weights 43000 and 25000. At the optimum pH 8.0 the specific activity of the purified enzyme is 630 units/mg protein, with L-T-glutamyl-pnitroanilide as substrate (K m : 0.9 raM) and 20 mM glycylgiycine as acceptor. The enzyme is inactivated by the active-site modifying agent and glutamine analogue, 6-diazo-5-oxo-L-norleucine, through a specific and stoichiometric reaction with the light subunit (K i : 1.2 mM); both the inactivation and the modification of the light subunit are accelerated by maleate and prevented by S-methylglutathione. The enzyme is also inactivated by the fluorescent alkylating agent 5-iodoacetamidofluorescein, by specific and stoichiometric incorporation of the fluorescent moiety into the light subunit, which is likewise prevented by S-methylglutathione, but is unaffected by maleate. Antiserum to rat kidney T-glutamyltransferase cross-reacts with the pancreas enzyme in immunodiffusion and inhibits its activity in the p-nitroanilide assay. Despite structural, enzymological and immunological similarities between the pancreas and kidney enzymes, their amino acid compositions are markedly different. The rat pancreas enzyme shows an interesting ontological development, being present in minimal amounts in the fetus, and increasing dramatically on birth and during the following 2 days.
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.
L-Asparaginase and L-Glutaminase: Sources, Production, and Applications in Medicine and Industry
Journal of microbiology, biotechnology and food sciences, 2019
Amidases (L-asparaginase and L-glutaminase) catalyze the deamination process of L-asparagine and L-glutamine to their corresponding acidic form with ammonia releasing. Both enzymes are considered one of the most biomedical and biotechnologically important groups of enzymes, besides their international contributing as an important commercial products. L-asparaginase and L-glutaminase have been receiving more attention as antileukemic agent for treatment of acute lymphoblastic leukemia (ALL) and other types of cancer. On the other hand, these enzymes also used in food manufacture for their hydrolysis effect and is a possible way to decrease the amount of free L-asparagine in the preliminary ingredients of food making, thus minimize the imminent risk of causing neurotoxic and carcinogenic acrylamide compound which formed when food heated above 120 °C. Glutamic and aspartic acid are important amino acids in food processing achieve a delicious, fine, sour and umami taste beside their nut...
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
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.