Modified assay procedure for enhanced sensitivity of in vitro glutamine synthetase (GS) activity measurements in marine phytoplankton

Clayton, JR; Ahmed, SI

doi:10.3354/meps036177

Cited by 6 publications

(2 citation statements)

References 10 publications

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“…the size of NH4P) using a Km value equivalent to 250 µM (at the high end of literature values (e.g. Syrett 1981;Clayton & Ahmed 1987;Ahmed & Hellebust 1988)). GS activity is also an inverse sigmoidal function of the GLN pool to achieve product inhibition and also a function of the availability of C (CGOGAT) for assimilation of inorganic N in the dark (equation 7.2).…”

Section: (D ) Assimilation Of Intracellular Ammonium and Growth (Figumentioning

confidence: 99%

Modelling the interactions between ammonium and nitrate uptake in marine phytoplankton

Flynn

Fasham

Hipkin

1997

Phil. Trans. R. Soc. Lond. B

131

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CONTENTS page 1. Introduction 1625 2. Theoretical bases and model development 1626 (a) Model construction 1626 (b) Control of transport (figures 2, 3; equation 5.x in table 5) 1629 (c) Nitrate reduction to ammonium (figures 2, 3; equation 6.x in table 6) 1630 (d) Assimilation of intracellular ammonium and growth (figures 2, 3; equation 7.x in table 7) 1633 (e) Operation in light-dark cycles 1635 (f) Parametrization 1635 3. Results 1635 (a) Simulation of ammonium-nitrate interactions 1635 (b) Sensitivity analysis 1637 4. Discussion 1639 References 1643 SUMMARY An empirically based mathematical model is presented which can simulate the major features of the interactions between ammonium and nitrate transport and assimilation in phytoplankton. The model (ammonium-nitrate interaction model), which is configured to simulate a generic microalga rather than a specified species, is constructed on simplified biochemical bases. A major requirement for parametrization is that the N:C ratio of the algae must be known and that transport and internal pool sizes need to be expressed per unit of cell C. The model uses the size of an internal pool of an early organic product of N assimilation (glutamine) to regulate rapid responses in ammoniumnitrate interactions. The synthesis of enzymes for the reduction of nitrate through to ammonium is induced by the size of the internal nitrate pool and repressed by the size of the glutamine pool. The assimilation of intracellular ammonium (into glutamine) is considered to be a constitutive process subjected to regulation by the size of the glutamine pool. Longer term responses have been linked to the nutrient history of the cell using the N:C cell quota. N assimilation in darkness is made a function of the amount of surplus C present and thus only occurs at low values of N:C. The model can simulate both qualitative and quantitative temporal shifts in the ammonium-nitrate interaction, while inclusion of a derivation of the standard quota model enables a concurrent simulation of cell growth and changes in nutrient status.

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Section: (D ) Assimilation Of Intracellular Ammonium and Growth (Figumentioning

confidence: 99%

Modelling the interactions between ammonium and nitrate uptake in marine phytoplankton

Flynn

Fasham

Hipkin

1997

Phil. Trans. R. Soc. Lond. B

131

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“…An alternative method, which overcomes these problems, is to measure the amount of Pi released during the reaction. This method has been used successfully to determine GS activity in a variety of organisms, including marine unicellular algae (Falkowski and Rivkin, 1976;Bressler and Ahmed, 1984;Clayton and Ahmed, 1987). Recent developments in the use of the malachite green assay for Pi have improved its stability and sensitivity (Baykov et al, 1988;Geladopoulos et al, 1991) and its use in measuring activities of enzyme reactions involving ATP hydrolysis (T.A.V.…”

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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)

Rees¹,

Larson²,

Heldens³

et al. 1995

Plant Physiol.

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~~ ~~~~~ ~~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-'

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Enzymes and Nitrogen Cycling

Berges¹,

Mulholland²

2008

Nitrogen in the Marine Environment

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Modified assay procedure for enhanced sensitivity of in vitro glutamine synthetase (GS) activity measurements in marine phytoplankton

Cited by 6 publications

References 10 publications

Modelling the interactions between ammonium and nitrate uptake in marine phytoplankton

Modelling the interactions between ammonium and nitrate uptake in marine phytoplankton

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)

Enzymes and Nitrogen Cycling

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