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Glutaminase Activity (glutaminase + activity)

Distribution by Scientific Domains

Chemistry	57%
Life Sciences	42%

Selected Abstracts

Effect of dexamethasone on neutrophil metabolism

CELL BIOCHEMISTRY AND FUNCTION, Issue 2 2003
Carolina Garcia
Abstract The effect of dexamethasone on glucose and glutamine metabolism was investigated. The consumption and oxidation of glucose and glutamine, and the production of glutamate and lactate were determined in neutrophils cultured for 3,h in the presence of dexamethasone. The activities and expression of glucose-6-phosphate dehydrogenase (G6PDH) and phosphate-dependent glutaminase were also determined under the same conditions. Addition of dexamethasone to the culture medium caused a significant increase of glucose consumption at 0.5,,m (123.9%) and 1.0,,m (78.3%) concentrations. In spite of this, however, glucose oxidation remained unchanged. The glucocorticoid did not change glutamine consumption but caused a significant increase of glutamate production and did not alter glutamine oxidation. Dexamethasone-treated neutrophils had a significant decrease of G6PDH activity and expression in particular at 1.0,,m concentration. Phosphate- dependent glutaminase activity was also decreased (about 34%) by dexamethasone treatment. A similar effect was observed on glutaminase expression as indicated by RT-PCR analysis. Thus, the effect of dexamethasone on neutrophil metabolism was particularly noticeable with respect to G6PDH and glutaminase activities where a decrease in the respective mRNA levels was demonstrated. Copyright © 2002 John Wiley & Sons, Ltd. [source]

Structural and functional insights into Erwinia carotovora l -asparaginase

FEBS JOURNAL, Issue 17 2008
Anastassios C. Papageorgiou
Bacterial l -asparaginases are enzymes that catalyze the hydrolysis of l -asparagine to aspartic acid. For the past 30 years, these enzymes have been used as therapeutic agents in the treatment of acute childhood lymphoblastic leukemia. Their intrinsic low-rate glutaminase activity, however, causes serious side-effects, including neurotoxicity, hepatitis, coagulopathy, and other dysfunctions. Erwinia carotovora asparaginase shows decreased glutaminase activity, so it is believed to have fewer side-effects in leukemia therapy. To gain detailed insights into the properties of E. carotovora asparaginase, combined crystallographic, thermal stability and cytotoxic experiments were performed. The crystal structure of E. carotovoral -asparaginase in the presence of l -Asp was determined at 2.5 Å resolution and refined to an Rcryst of 19.2 (Rfree = 26.6%) with good stereochemistry. Cytotoxicity measurements revealed that E. carotovora asparaginase is 30 times less toxic than the Escherichia coli enzyme against human leukemia cell lines. Moreover, denaturing experiments showed that E. carotovora asparaginase has decreased thermodynamic stability as compared to the E. coli enzyme and is rapidly inactivated in the presence of urea. On the basis of these results, we propose that E. carotovora asparaginase has limited potential as an antileukemic drug, despite its promising low glutaminase activity. Our analysis may be applicable to the therapeutic evaluation of other asparaginases as well. [source]

Characterization of the products of the genes SNO1 and SNZ1 involved in pyridoxine synthesis in Saccharomyces cerevisiae

FEBS JOURNAL, Issue 4 2004
Yi-Xin Dong
Genes SNO1 and SNZ1 are Saccharomyces cerevisiae homologues of PDX2 and PDX1 which participate in pyridoxine synthesis in the fungus Cercospora nicotianae. In order to clarify their function, the two genes SNO1 and SNZ1 were expressed in Escherichia coli either individually or simultaneously and with or without a His-tag. When expressed simultaneously, the two protein products formed a complex and showed glutaminase activity. When purified to homogeneity, the complex exhibited a specific activity of 480 nmol·mg,1·min,1 as glutaminase, with a Km of 3.4 mm for glutamine. These values are comparable to those for other glutamine amidotransferases. In addition, the glutaminase activity was impaired by 6-diazo-5-oxo- l -norleucine in a time- and dose-dependent manner and the enzyme was protected from deactivation by glutamine. These data suggest strongly that the complex of Sno1p and Snz1p is a glutamine amidotransferase with the former serving as the glutaminase, although the activity was barely detectable with Sno1p alone. The function of Snz1p and the amido acceptor for ammonia remain to be identified. [source]

Long-range allosteric transitions in carbamoyl phosphate synthetase

PROTEIN SCIENCE, Issue 9 2004
James B. Thoden
Abstract Carbamoyl phosphate synthetase plays a key role in both pyrimidine and arginine biosynthesis by catalyzing the production of carbamoyl phosphate from one molecule of bicarbonate, two molecules of MgATP, and one molecule of glutamine. The enzyme from Escherichia coli consists of two polypeptide chains referred to as the small and large subunits, which contain a total of three separate active sites that are connected by an intramolecular tunnel. The small subunit harbors one of these active sites and is responsible for the hydrolysis of glutamine to glutamate and ammonia. The large subunit binds the two required molecules of MgATP and is involved in assembling the final product. Compounds such as L-ornithine, UMP, and IMP allosterically regulate the enzyme. Here, we report the three-dimensional structure of a site-directed mutant protein of carbamoyl phosphate synthetase from E. coli, where Cys 248 in the small subunit was changed to an aspartate. This residue was targeted for a structural investigation because previous studies demonstrated that the partial glutaminase activity of the C248D mutant protein was increased 40-fold relative to the wild-type enzyme, whereas the formation of carbamoyl phosphate using glutamine as a nitrogen source was completely abolished. Remarkably, although Cys 248 in the small subunit is located at ,100 Å from the allosteric binding pocket in the large subunit, the electron density map clearly revealed the presence of UMP, although this ligand was never included in the purification or crystallization schemes. The manner in which UMP binds to carbamoyl phosphate synthetase is described. [source]

Structure of Helicobacter pyloril -asparaginase at 1.4,Å resolution

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 12 2009
Prathusha Dhavala
Bacterial l -asparaginases have been used in the treatment of childhood acute lymphoblastic leukaemia for over 30,years. Their therapeutic effect is based on their ability to catalyze the conversion of l -asparagine, an essential amino acid in certain tumours, to l -aspartic acid and ammonia. Two l -asparaginases, one from Escherichia coli and the other from Erwinia chrysanthemi, have been widely employed in clinical practice as anti-leukaemia drugs. However, l -asparaginases are also able to cause severe side effects owing to their intrinsic glutaminase activity. Helicobacter pyloril -asparaginase (HpA) has been reported to have negligible glutaminase activity. To gain insight into the properties of HpA, its crystal structure in the presence of l -aspartate was determined to 1.4,Å resolution, which is one of the highest resolutions obtained for an l -asparaginase structure. The final structure has an Rcryst of 12.6% (Rfree = 16.9%) with good stereochemistry. A detailed analysis of the active site showed major differences in the active-site flexible loop and in the 286,297 loop from the second subunit, which is involved in active-site formation. Accordingly, Glu289, Asn255 and Gln63 are suggested to play roles in modulating the accessibility of the active site. Overall, the structural comparison revealed that HpA has greater structural similarity to E. colil -asparaginase than to any other l -asparaginase, including Er. carotovoral -asparaginase, despite the fact that the latter is also characterized by low glutaminase activity. [source]

Measurement of key metabolic enzyme activities in mammalian cells using rapid and sensitive microplate-based assays

BIOTECHNOLOGY & BIOENGINEERING, Issue 3 2010
R. Janke
Abstract Sensitive microplate-based assays to determine low levels of key enzyme activities in mammalian cells are presented. The enzyme platform consists of four cycling assays to measure the activity of 28 enzymes involved in central carbon and glutamine metabolism. The sensitivity limit of all cycling assays was between 0.025 and 0.4,nmol product. For the detection of glutaminase activity, a new glutamate cycle system involving the enzymes glutamate dehydrogenase and aspartate transaminase was established. The relative standard deviation of the method was found to be 1.7% with a limit of detection of 8.2,pmol and a limit of quantitation of 24.8,pmol. Hence, cell extracts could be highly diluted to reduce interferences caused by other components in the extract, which in addition minimized underestimates or overestimates of actual enzyme activities. Since substrate concentrations could be maintained at a nearly constant level throughout the assay product accumulation during the reaction was low, which minimized product inhibition. As an example, the enzyme platform was used to investigate maximum enzyme activities of stationary-phase MDCK cells grown in serum-containing GMEM medium as typically used in influenza vaccine production. Biotechnol. Bioeng. 2010;107: 566,581. © 2010 Wiley Periodicals, Inc. [source]