In situ glutamine synthetase activity in a marine unicellular alga: development of a sensitive colorimetric assay and the effects of nitrogen status on enzyme activity

• Published in: Plant physiology (Bethesda) 1995-12, Vol.109 (4), p.1405-1410
• Main Authors: Rees, T.A.V. (University of Auckland, Warkworth, New Zealand.), Larson, T.R, Heldens, J.W.G, Huning, F.G.J
• Format: Article
• Language: English
• Subjects: ABSORCION DE SUSTANCIAS NUTRITIVAS; ABSORPTION DE SUBSTANCES NUTRITIVES; ACIDE GLUTAMIQUE; ACIDO GLUTAMICO; ACTIVIDAD ENZIMATICA; ACTIVITE ENZYMATIQUE; ADENOSINE TRIPHOSPHATE; ADENOSINTRIFOSFATO; Algae; AMINAS; AMINE; Ammonia; AMMONIAC; AMONIACO; AZOTE; BACILLARIOPHYCEAE; Biochemistry and Enzymology; Biological and medical sciences; BIOSINTESIS; BIOSYNTHESE; Chemical suspensions; CHLOROPHYLLE; CICLO DEL NITROGENO; CLOROFILAS; COMPOSE DE L'AMMONIUM; COMPUESTOS DE AMONIO; CULTIVO DE CELULAS; CULTURE DE CELLULE; CYCLE DE L'AZOTE; Diatoms; DISPONIBILIDAD DE NUTRIENTES; DISPONIBILITE D'ELEMENT NUTRITIF; DOSAGE BIOLOGIQUE; ENSAYO BIOLOGICO; Enzyme activity; Enzymes; Fundamental and applied biological sciences. Psychology; MEDIO DE CULTIVO; Metabolism; Methylamines; MILIEU DE CULTURE; Nitrogen; NITROGENO; Phytoplankton; Plant physiology and development; Quaternary ammonium compounds; REDUCCION; REDUCTION; Starvation; TEMPERATURA; TEMPERATURE
• ISSN: 0032-0889; 1532-2548
• DOI: 10.1104/pp.109.4.1405

A malachite green colorimetric assay for glutamine synthetase is described. Glutamine synthetase activity was determined in situ in the marine diatom Phaeodactylum tricornutum Bohlin using cells permeabilized by freeze/thawing. Higher activities were obtained with cells permeabilized in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid compared with N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid, tris(hydroxymethyl)aminomethane, or imidazole, and the optimum pH was 7.9. Activities were higher in cells permeabilized in the presence of reductant, particularly dithiothreitol. Glutamine synthetase activities were markedly decreased in the presence of methionine sulfoximine. In the presence of saturating concentrations of glutamate and ATP, the apparent Km for ammonia was 320 micromolar, but this value decreased to 110 micromolar with subsaturating concentrations of glutamate and ATP. The apparent Km values for glutamate and ATP, in the presence of saturating concentrations of ammonia, were 9.7 and 2.9 mM, respectively. Ammonia-grown cells had lower glutamine synthetase activities than did nitrate-grown cells. During nitrogen starvation of both ammonia- and nitrate-grown cells, glutamine synthetase activities increased rapidly during the first 8 h, reaching maximum values after 24 to 48 h. Moreover, the time course for the increases in glutamine synthetase activities and rate of methylamine uptake following the transfer of nitrate-grown cells to nitrogen-deficient medium were very similar. In nitrate-grown cells and cells deprived of combined nitrogen, glutamine synthetase activities and maximum rates of ammonia uptake gave comparable values when measured at the same temperature (20 degrees C)