University of North Florida
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Stuart Chalk, Ph.D.
Department of Chemistry
University of North Florida
Phone: 1-904-620-1938
Fax: 1-904-620-3535
Website: @unf

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Fish Retina

Classification: Biological tissue -> retina -> fish

Citations 1

"High Performance Liquid Chromatography Assay For Glutamine Synthetase"
Neurochem. Int. 1989 Volume 14, Issue 4 Pages 491-496
S. Kato,*, K. Negishi, K. Honma, K. Sakai and Y. Shimada

Abstract: Cultured fish retina and clonal neural cell lines (rat C6 glioma, mouse clonal NIE 115 and N18TG2 neuroblastoma) were homogenized in 10 mM imidazole hydrochloride buffer (pH 7.2) containing 0.5 mM EDTA, 5 mM 2-mercaptoethanol and 0.5 mM phenylmethylsulfonyl fluoride. Glutamate-ammonia ligase (I) in the cell-free extracts was assayed in a reaction mixture (total volume 1 mL) containing 100 mM imidazole hydrochloride buffer (pH 7.2), 20 mM MgCl2, 25 mM 2-mercaptoethanol, 20 mM Na L-glutamate, 50 mM hydroxylamine, pyruvate kinase, 10 mM phosphoenolpyruvate trichlorohexamine and 20 mM ATP. After incubation at 25°C (for fish retina) or 37°C (for clonal cells), the reaction was stopped with 2.5% sulfosalicylic acid (50 µL) and the mixture was centrifuged and filtered. The γ-glutamyl hydroxamate formed was determined by HPLC using a Hitachi 2619F ion-exchange column (15 cm x 4 mm) with post-column derivatization with phthalaldehyde. From 50 to 500 pM-I could be determined by this method compared to 100 to 200 nM-I by the conventional colorimetric method (Rowe et al., Methods in Enzymology, 1970, 17, 900).
Enzyme, glutamate synthetase HPLC Fluorescence Buffer Heated reaction Post-column derivatization Method comparison