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)

Abstract

~~ ~~~~~ ~~A malachite green colorimetric assay for glutamine synthetase i s described. Glutamine synthetase activity was determined in situ in the marine diatom Phaeodacfylum fricornufum Bohlin using cells permeabilized by freeze/thawing. Higher activities were obtained with cells permeabilized in N-2-hydroxyethylpiperazine-Nf-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. Clutamine synthetase activities were markedly decreased in the presence of methionine sulfoximine. I n the presence of saturating concentrations of glutamate and ATP, the apparent K,,, for ammonia was 320 WM, but this value decreased to 110 p~ with subsaturating concentrations of glutamate and ATP. The apparent K,,, 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 t o 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. I n 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 (20OC).Marine unicellular algae (phytoplankton) are responsible for about 70% of global primary nitrogen assimilation (Raven et al., 1993). Despite the importance of marine unicellular algae in the global nitrogen cycle and the compelling evidence that GS is the primary ammonia-assimilating enzyme in plants (Miflin and Lea, 1980), there is remarkably little information concerning GS in marine uni-'

Yield and biosynthesis of nitrogenous compounds in fruits of green bean (Phaseolus vulgaris L cv Strike) in response to increasing N fertilisation

“…The extraction of glutamine synthetase followed the method of Rees et al 21 The assay was based on that described by Slawyk and Rodier. 22 The reaction mixture contained 0.2 M Hepes-KOH, pH 7.9, 50 mM MgCl 2 , 5 mM Na 2 EDTA, 100 mM potassium glutamate, 50 mM ammonium acetate and 8 mM ATP.…”

Nitrogen status and metabolism in the green seaweed Enteromorpha intestinalis: an examination of three natural populations

“…Glutamine synthetase activity was determined as described previously (Rees et al 1995 at 17.5°C. Rates were normalised to dry weight using a dry weight:fresh weight ratio of 0.25.…”

Nitrogen assimilation characteristics of polar seaweeds from differing nutrient environments

“…GS activity was measured foilowing the method of Rees et al (1995) in an assay mix containing 50 p1 crude enzyme extract, 8 mM ATP, 100 mM glutamate. and 50 mM ammonium chloride added to basic HEPES buffer (minus PVP and Triton-X) to give a final volume of 750 p1.…”

“…Samples were incubated for 15 min at 30°C, the reaction stopped by adding 0.25 ml 1 M H2SO4, and the samples centnfuged for 1 min (16000 X g ) . Of the resulting supernatant, 25 pl was added to 975 pl of distilled water, and inorganic phosphate was measured as in Rees et al (1995).…”