Athwal, Gurdeep Singh; (1995) Glutamate dehydrogenase, its role, regulation and characterisation in carrot cell suspension cultures. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The enzyme glutamate dehydrogenase (GDH) occupies a position in plant biochemistry which links carbon and nitrogen metabolism, via the anabolism or catabolism of glutamate. However, its role in higher plants has not been fully resolved. Cell suspension cultures of Daucus carota were used to investigate the possible role, regulation and characterisation of GDH in higher plants. GDH activity was seen to be indirectly regulated by the carbon source, and its availability. The addition of tricarboxylic acid cycle intermediates were found to not significantly affect GDH activity in both repressed and de-repressed cell suspension cultures. However, the nucleotide adenosine 3',5'-cyclic monophosphate was shown to cause de-repression in both carbon stressed, and non-stressed cultures, which suggested a possible regulatory mechanism akin to prokaryotic catabolite repression. Additions of other adenosine nucleotides to the cultures also suggested a possible relationship between adenylate concentrations and GDH activity. The enzyme was found to be neither regulated by ammonium or directly involved in ammonium assimilation. Using electron transport and oxidative phosphorylation inhibitors a correlation between respiratory control and de-repression of GDH was established. The function of GDH appeared to be related to glutamate catabolism, caused indirectly by carbon stress, and directly by energy stress. The enzyme was purified to homogeneity and found to have similar physical properties and characteristics to other plant GDHs. KM values obtained were similar to other reported values, and confirmed the relatively high KM for ammonium, compared to glutamine synthetase. N-terminal end sequence data was obtained, and found to show no homology to other eukaryotic and prokaryotic GDH protein sequences. Using protein synthesis inhibitors it was found to be nuclear coded. Both subunit and native molecular weights were determined, and suggested that the enzyme had a hexameric structure, being possibly composed of four or five distinct subunits, with similar molecular weights, and electrophoretic properties. At least ten active deaminating isoforms were isolated, which were not a result of somaclonal variation.

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