Protocols to assay the activity of glutamine synthetase (GS) are presented as they have been used in our laboratory to correlate the expression levels of the gene encoding Drosophila GS1 gene, the GS1 protein levels, and its activity in extracts of larvae and heads from Drosophila melanogaster. The assays are based on the glutamine synthetase-catalyzed formation of γ-glutamylhydroxylamine in the presence of ATP, L-glutamate, and hydroxylamine, in which hydroxylamine substitutes for ammonia in the reaction. Formation of γ-glutamylhydroxylamine is monitored spectrophotometrically in discontinuous assays upon complex formation with FeCl3. Fixed-time assays and those based on monitoring the time-course of product formation at different reaction times are described. The protocols can be adapted to quantify glutamine synthetase activity on biological materials other than Drosophila.
Quantitation of Glutamine Synthetase 1 Activity in Drosophila melanogaster / T. Vitali, M.A. Vanoni, P. Bellosta (METHODS IN MOLECULAR BIOLOGY). - In: Metobolic Reprogramming / [a cura di] S. Papa. - [s.l] : Humana Press, 2023. - ISBN 978-1-0716-3246-8. - pp. 237-260 [10.1007/978-1-0716-3247-5_18]
Quantitation of Glutamine Synthetase 1 Activity in Drosophila melanogaster
T. VitaliPrimo
Writing – Review & Editing
;M.A. Vanoni
Secondo
Writing – Review & Editing
;P. Bellosta
Ultimo
Writing – Review & Editing
2023
Abstract
Protocols to assay the activity of glutamine synthetase (GS) are presented as they have been used in our laboratory to correlate the expression levels of the gene encoding Drosophila GS1 gene, the GS1 protein levels, and its activity in extracts of larvae and heads from Drosophila melanogaster. The assays are based on the glutamine synthetase-catalyzed formation of γ-glutamylhydroxylamine in the presence of ATP, L-glutamate, and hydroxylamine, in which hydroxylamine substitutes for ammonia in the reaction. Formation of γ-glutamylhydroxylamine is monitored spectrophotometrically in discontinuous assays upon complex formation with FeCl3. Fixed-time assays and those based on monitoring the time-course of product formation at different reaction times are described. The protocols can be adapted to quantify glutamine synthetase activity on biological materials other than Drosophila.File | Dimensione | Formato | |
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