A high performance liquid chromatography assay for glutamine synthetase.
A sensitive method for assaying glutamine synthetase activity is described. This enzyme produces ?-glutamylhydroxamate in the presence of l-glutamic acid and hydroxylamine as substrates. This amino acid hydroxamate was separated and quantified by high performance liquid chromatography on an ion-exchange resin column using post-column derivatization with o-phthalaldehyde for detection. As little as 50 pmol of ?-glutamylhydroxamate was detected in assays using cell-free extracts from fish retina, rat clonal C6 glioma cells, mouse clonal NIE 115 and N18TG2 neuroblastoma cells, whose specific activity measured was 1.0, 0.03, 0.01 and 0.01 ?mol of ?-glutamylhydroxamate produced per 30 min per milligram protein, respectively.
Kato
S
S
Molecular Neurobiology Group, Department of Neurophysiology, Neuroinformation Research Institute (NIRI), School of Medicine, University of Kanazawa, Kanazawa, Ishikawa 920, Japan.
Negishi
K
K
Honma
K
K
Sakai
K
K
Shimada
Y
Y