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

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 K-m for ammonia was 320 mu M, but this value decreased to 110 mu M with subsaturating concentrations of glutamate and ATP. The apparent K-m 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).