Ammonia assimilating enzymes from cyanobacteria: in situ and in vitro assay using high-performance liquid chromatography.
Assay systems for ammonia assimilating enzymes in cyanobacteria are reported. Glutamine synthetase, glutamate synthase, and glutamate dehydrogenase can be easily assayed in situ, after the cells are made permeable to the reagents, or in vitro. The method is based upon the quantitation of glutamine or glutamate after the separation, when needed, of their o-phthaldialdehyde derivatives by reverse-phase high-performance liquid chromatography on a C18 column. The isocratic elution and the fluorometric detection of the amino acid derivatives make the method fast, simple, sensitive, and free of the assay artifacts which can be produced in coupled assays or when spectrophotometric measurements are carried out in the turbid samples employed for in situ assays.
Marqués
S
S
Departamento de Bioquímica Vegetal y Biología Molecular, Universidad de Sevilla, Spain.
Florencio
F J
FJ
Candau
P
P
D000641:Ammonia / Q000378:metabolism*; D001322:Autoanalysis; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D000458:Cyanobacteria / Q000201:enzymology*; D005969:Glutamate Dehydrogenase / Q000378:metabolism*; D005970:Glutamate Synthase / Q000378:metabolism*; D005974:Glutamate-Ammonia Ligase / Q000378:metabolism*; D007700:Kinetics; D013050:Spectrometry, Fluorescence; D000637:Transaminases / Q000378:metabolism*
D000641:Ammonia; D005970:Glutamate Synthase; D005969:Glutamate Dehydrogenase; D000637:Transaminases; D005974:Glutamate-Ammonia Ligase