A rapid radioactive assay for glutamine synthetase, glutaminase, asparagine synthetase, and asparaginase

Prusiner, Stanley B.; Milner, Larry

doi:10.1016/0003-2697(70)90069-2

Cited by 133 publications

(43 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Transferase activity was measured by the y-glutamyl transferase reaction (Ferguson & Sims, 1971). One unit of enzyme activity is defined as the formation of 1 pmol yglutamylhydroxamate min-' at 37 "C. Biosynthetic activity was measured either according to Bender et al (1977), except that cetyltrimethylammonium bromide was omitted, or with the coupled assay system described by Kingdon et al (1968), or by the formation of radioactive glutamine, as described by Prusiner & Milner (1970). In the first assay, y-glutamylhydroxamate is measured using glutamate in the reaction mixture and the unit of activity is defined as in the case of the transferase reaction.…”

Section: Methodsmentioning

confidence: 99%

Dissociation by NH4Cl treatment of the enzymic activities of glutamine synthetase II from Rhizobium leguminosarum biovar viceae

Manco

Rossi²,

Defez³

et al. 1992

Journal of General Microbiology

View full text Add to dashboard Cite

Glutamine synthetase I1 (GSII) was purified to homogeneity from Rhizobium leguminosarum biovar viceae and characterized.-The Sequence of 26 m i n o acid?esidues from the amino-terminal end bf the protein showed high similarity with the sequence of GSII from Bradyrhizobium japonicum or from Rhizobium meliloti. Non-denaturing PAGE showed that GSII, either in crude extracts or in the pure state, was a mixture of an octamer and a tetramer and that under specific conditions the octamerltetramer ratio could be modified in either direction. The pure enzyme was used to raise an antiserum which was highly specific. Addition of NH4CI to a bacterial culture derepressed for GSII caused a specific decrease in transferase activity, faster than the one observed when the amount of immunoreactive material was measured by different methods. On the other hand, biosynthetic activity, measured as the rate of ADP or glutamine formation, paralleled the rate of decrease in immunoreactive material. A partially purified enzyme preparation retained this dissociation of kinetic parameters, strongly suggesting a post-translational modification. These findings are discussed with respect to the possible role of GSII in the Rhizobium-legume symbiosis.

show abstract

Section: Methodsmentioning

confidence: 99%

Dissociation by NH4Cl treatment of the enzymic activities of glutamine synthetase II from Rhizobium leguminosarum biovar viceae

Manco

Rossi²,

Defez³

et al. 1992

Journal of General Microbiology

View full text Add to dashboard Cite

show abstract

“…GLNase, which is predominantly localized between outer and inner mitochondrial membranes, is regulated by local glutamine availability but also by the levels of intracellular inorganic phosphate N-acetyl-aspartate, taurine, histidine, lysine, and intermediates of TCA (29,36). The previously developed methods were optimized for isolated enzymes or homogenates of cells and tissues (25,27). However, using isolated enzymes removes the intrinsic regulation of GS and GLNase in a "native" environment.…”

Section: Discussionmentioning

confidence: 99%

“…This assay was designed based on the method proposed by Prusiner and Milner (27) that involves separation of radiolabeled amino acids on Dowex anion exchange columns. However, the original method was designed for use with isolated enzymes and did not take into account the possibility that both the substrate and the product of the GS reaction may be consumed in alternative enzymatic processes.…”

Section: Effects Of the Inhibitor Of Glutamine Synthetase Mso And Thementioning

confidence: 99%

“…However, such an approach requires sophisticated equipment and data processing. More accessible methodology for accurate measurements of GS and GLNase activities involves quantification of the enzymatic conversion of 3 H-or 14 C-labeled amino acids (25,27). Yet, the existing radiolabeled amino acid assays were developed for purified proteins or tissue homogenates.…”

mentioning

confidence: 99%

See 1 more Smart Citation

A simple method for measuring intracellular activities of glutamine synthetase and glutaminase in glial cells

Mongin

Hyzinski‐García

Vincent

et al. 2011

American Journal of Physiology-Cell Physiology

View full text Add to dashboard Cite

Mongin AA, Hyzinski-García MC, Vincent MY, Keller RW Jr. A simple method for measuring intracellular activities of glutamine synthetase and glutaminase in glial cells. Am J Physiol Cell Physiol 301: C814-C822, 2011. First published July 6, 2011; doi:10.1152/ajpcell.00035.2011.-Here we report and validate a simple method for measuring intracellular activities of glial glutamine synthetase (GS) and glutaminase (GLNase) in intact glial cells. These enzymes are responsible for glutamate and glutamine recycling in the brain, where glutamate and glutamine transport from the blood stream is strongly limited by the blood-brain barrier. The intracellular levels of glutamate and glutamine are dependent on activities of numerous enzymatic processes, including 1) cytosolic production of glutamine from glutamate by GS, 2) production of glutamate from glutamine by GLNase that is primarily localized between mitochondrial membranes, and 3) mitochondrial conversion of glutamate to the tricarboxylic cycle intermediate ␣-ketoglutarate in the reactions of oxidative deamination and transamination. We measured intracellular activities of GS and GLNase by quantifying enzymatic interconversions of L-[ 3 H]glutamate and L-[ 3 H]glutamine in cultured rat astrocytes. The intracellular substrate and the products of enzymatic reactions were separated in one step using commercially available anion exchange columns and quantified using a scintillation counter. The involvement of GS and GLNase in the conversion of 3 H-labeled substrates was verified using irreversible pharmacological inhibitors for each of the enzymes and additionally validated by measuring intracellular amino acid levels using an HPLC. Overall, this paper describes optimized conditions and pharmacological controls for measuring GS and GLNase activities in intact glial cells.glutamate-glutamine cycle; metabolism; astrocytes L-GLUTAMATE and L-glutamine are two major intermediates of nitrogen metabolism. These two amino acids are present at high micromolar (ϳ30 -500 M) levels in the circulatory system. They diffuse freely from capillaries to the interstitial liquids and extracellular space and are taken inside animal cells by several types of amino acid transporters (24). In cytosol and mitochondrial matrix, concentrations of glutamate and glutamine are in the millimolar range. These two amino acids exist in equilibrium because they are consumed and produced in numerous interrelated biochemical reactions. The abundance and the generic roles of glutamate and glutamine in metabolism and protein synthesis interfere with the signaling properties of glutamate in the central nervous system (CNS). In the brain, glutamate serves as the main excitatory neurotransmitter. Therefore, extracellular levels of glutamate have to be tightly regulated and kept at low levels (reviewed in Refs. 9 and 14). This is possible due to two major factors: 1) limited transport of glutamate and glutamine via the endothelial blood-brain barrier (33), and 2) continuous recycling of glutamate and glutamine in t...

show abstract

“…and GOGAT (EC 1.4.1.14) activities were measured spectrophotometrically as described by Groat and Vance (10). GS (EC 6.3.1.2), activity was measured radiometrically according to Prusiner and Milner (20). GOT (EC 2.6.1.1) activity was measured according to Ryan et al (23).…”

Section: Methodsmentioning

confidence: 99%

Tissue Cultures Derived from Ineffective Root Nodules of Alfalfa

Vance

Johnson

Boylan³

1984

Plant Physiol.

View full text Add to dashboard Cite

show abstract

A rapid radioactive assay for glutamine synthetase, glutaminase, asparagine synthetase, and asparaginase

Cited by 133 publications

References 27 publications

Dissociation by NH4Cl treatment of the enzymic activities of glutamine synthetase II from Rhizobium leguminosarum biovar viceae

Dissociation by NH4Cl treatment of the enzymic activities of glutamine synthetase II from Rhizobium leguminosarum biovar viceae

A simple method for measuring intracellular activities of glutamine synthetase and glutaminase in glial cells

Tissue Cultures Derived from Ineffective Root Nodules of Alfalfa

Contact Info

Product

Resources

About