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Enzymes of ammonia assimilation in lupin root nodules and callus cells

Ling, Ping Siong (1980) Enzymes of ammonia assimilation in lupin root nodules and callus cells. PhD thesis, Murdoch University.

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Glutamate synthase (EC. and has been shown present in a number of legume root nodules, partly in bacteroids but mainly in the plant cytosol fraction. Isolation of this enzyme from the plant fraction of legume nodules required dithiothreitol or mercaptoethanol to obtain activity of the enzyme. Glutamate synthase from broad bean bacteroid is specific for NADPH, but the nodule cytosol enzyme is active with NADH. The levels of activity measurable in the nodule supernatant preparations are dependent on the concentrations of mercaptoethanol and EDTA used in the extraction medium, highest activity being obtained with imidazole-HCL buffer (pH 7.8) containing 0.14 M mercaptoethanol and 3 mM EDTA.

The lupin nodule cytosol glutamate synthase was labile and required protection by high levels of thiols; glycerol and the substrates also stabilized it. It had a molecular weight of 220,000 and did not appear to contain any metal. Electrophoresis of the purified enzyme on sodium dodecyl sulphate polyacrylamide gel gave three protein bands corresponding to molecular weight of 39,800; 37,600 and 32,000. The optimum pH was 8.25, at which Km values for 2-oxoglutarate, glutamine and NADH were 0.02 mM, 0.32 mM and 0.01 mM respectively. Activity was inhibited by NAD+ and several amino acids. Glutamine protected it from inhibition by aminobutyric acid, citrulline, methionine, proline, serine and threonine. Several metabolites, metals and monovalent cations were also tested for possible effects on enzyme activity. Several monovalent cations were stimulatory, whereas most heavy metals were inhibitory.

The free amino acid pools in the plant and bacteroid fractions of lupin and broad bean nodules contain a variety of amino acids with asparagine, glutamate, aspartate and alanine being present in much higher concentrations than the others. Comparisons between the concentrations of various amino acids in the plant fraction of lupin nodules and their inhibitory patterns indicate that, apart possibly from aminobutyrate and glutamate product inhibition, amino acids play little role in the regulation of glutamate synthase activity.

Studies on the development of nitrogenase (EC. and ammonia assimilatory enzymes in lupin nodules showed that the specific and total activities of nitrogenase, plant glutamate synthase, plant glutamine synthetase (EC., plant glutamate dehydrogenase (EC. and plant alanine dehydrogenase (EC. increased rapidly during nodule development. During active nitrogen fixation, the total activities of glutamine synthetase and glutamate synthase decreased, but glutamate and alanine dehydrogenase increased. When nitrogenase activity decreased during pod formation and filling, the total activities of glutamate and alanine dehydrogenases decreased rapidly, but glutamine synthetase and glutamate synthase increased. The specific activities of the bacteroid ammonia assimilatory enzymes varied only slightly throughout the plants' growth cycle, and were inadequate to account for the observed rates of nitrogen fixation in intact nodules. The total activity of the plant glutamine synthetase, except during active nitrogen fixation, was higher than that of nitrogenase, while that of the plant glutamate dehydrogenase was higher than that of nitrogenase throughout the nodules' function. The total activity of the plant alanine dehydrogenase was comparable with that of nitrogenase during the period of active nitrogen fixation. It was absent in the ineffective nodules.

Partially purified glutamine synthetase, glutamate and alanine dehydrogenases from lupin nodule cytosol had apparent Km values of 0.14 mM, 26.3 mM and 6.02 mM for ammonia, respectively. Ammonia concentrations found in the plant fraction of a number of legume root nodules were between 4.5 and 5 mM during active nitrogen fixation. Concentrations between 5.5 and 6 mM were measured in various bacteroids; these values very likely are conservative.

Increasing ammonia availability leads to a decrease in level of glutamine synthetase and glutamate synthase and an increase in the level of glutamate dehydrogenase in lucerne cell suspension cultures, and vice versa. Analysis of ammonia showed that these changes in enzyme level were accompanied by changes in the pool ammonia level. Regardless of whether ammonium, nitrate, or glutamine was supplied as the nitrogen source for growth, glutamine synthetase and glutamate synthase activities were highest when the internal ammonia concentrations were between 1.5 and 1.8 mM. Cultures supplemented with amino acids had lower levels of glutamine synthetase and glutamate synthase and higher intracellular ammonia concentration than the control.

An overall scheme for the integration of these enzymes in the assimilation of ammonia is presented. It is proposed that ammonia produced by nitrogen fixation is assimilated mainly outside the bacteroids and that the plant glutamine synthetase-glutamate synthase system, glutamate and alanine dehydrogenases play an important assimilatory role at different periods of nodule function.

Item Type: Thesis (PhD)
Murdoch Affiliation: School of Environmental and Life Sciences
Notes: Note to the author: If you would like to make your thesis openly available on Murdoch University Library's Research Repository, please contact: Thank you.
Supervisor(s): Dilworth, Michael
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