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 descr ibed. Glutamine synthetase activity was determined in situ in the mari ne diatom Phaeodactylum tricornutum Bohlin using cells permeabilized b y freeze/thawing. Higher activities were obtained with cells permeabil ized in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid compared w ith N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid, tris(hydr oxymethyl)aminomethane, or imidazole, and the optimum pH was 7.9. Acti vities were higher in cells permeabilized in the presence of reductant , particularly dithiothreitol. Glutamine synthetase activities were ma rkedly decreased in the presence of methionine sulfoximine. In the pre sence 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 wit h subsaturating concentrations of glutamate and ATP. The apparent K-m values for glutamate and ATP, in the presence of saturating concentrat ions of ammonia, were 9.7 and 2.9 mM, respectively. Ammonia-grown cell s had lower glutamine synthetase activities than did nitrate-grown cel ls. During nitrogen starvation of both ammonia- and nitrate-grown cell s, glutamine synthetase activities increased rapidly during the first 8 h, reaching maximum values after 24 to 48 h. Moreover, the time cour se for the increases in glutamine synthetase activities and rate of me thylamine uptake following the transfer of nitrate-grown cells to nitr ogen-deficient medium were very similar. In nitrate-grown cells and ce lls 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).