Purification of Mitochondrial Glutamate Dehydrogenase from Dark-Grown Soybean Seedlings

Turano, Frank J.; Dashner, Ralph; Upadhyaya, Abha; Caldwell, Charles R.

doi:10.1104/pp.112.3.1357

Cited by 97 publications

(74 citation statements)

References 26 publications

(23 reference statements)

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“…All extractions were performed at 4°C. GDH was measured as described by Turano et al (1996) except that the extraction buffer was the same as for GS (Tercé-Laforgue et al, 2004). Soluble protein was determined using a commercially available kit (Coomassie Protein assay reagent, Bio-Rad, Munich, Germany) using bovine serum albumin (BSA) as a standard.…”

Section: Enzymatic Assay and Determination Of Total Soluble Proteinmentioning

confidence: 99%

Glutamate Dehydrogenase of Tobacco Is Mainly Induced in the Cytosol of Phloem Companion Cells When Ammonia Is Provided Either Externally or Released during Photorespiration

et al. 2004

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Glutamate (Glu) dehydrogenase (GDH) catalyses the reversible amination of 2-oxoglutarate for the synthesis of Glu using ammonium as a substrate. This enzyme preferentially occurs in the mitochondria of companion cells of a number of plant species grown on nitrate as the sole nitrogen source. For a better understanding of the controversial role of GDH either in ammonium assimilation or in the supply of 2-oxoglutarate (F. Dubois, T. Tercé-Laforgue, M.B. Gonzalez-Moro, M.B. Estavillo, R. Sangwan, A. Gallais, B. Hirel [2003] Plant Physiol Biochem 41: 565-576), we studied the localization of GDH in untransformed tobacco (Nicotiana tabacum) plants grown either on low nitrate or on ammonium and in ferredoxin-dependent Glu synthase antisense plants. Production of GDH and its activity were strongly induced when plants were grown on ammonium as the sole nitrogen source. The induction mainly occurred in highly vascularized organs such as stems and midribs and was likely to be due to accumulation of phloem-translocated ammonium in the sap. GDH induction occurred when ammonia was applied externally to untransformed control plants or resulted from photorespiratory activity in transgenic plants down-regulated for ferredoxin-dependent Glu synthase. GDH was increased in the mitochondria and appeared in the cytosol of companion cells. Taken together, our results suggest that the enzyme plays a dual role in companion cells, either in the mitochondria when mineral nitrogen availability is low or in the cytosol when ammonium concentration increases above a certain threshold.Ammonium is the ultimate form of inorganic nitrogen available to the plant. It can originate from a wide variety of metabolic processes such as nitrate reduction, photorespiration, phenylpropanoid metabolism, utilization of nitrogen transport compounds, amino acid catabolism, symbiotic nitrogen fixation (Hirel and Lea, 2001), and insect digestion in carnivorous plants (Schulze et al., 1997). It is then incorporated into an organic molecule, 2-oxoglutarate, by the combined action of the enzymes Gln synthetase (GS) and Glu synthase (Gln-2-oxoglutarate aminotransferase; GOGAT) to allow the synthesis of Gln and Glu. Both amino acids are further used as amino group donors for the synthesis of virtually all the nitrogencontaining molecules including the other amino acids needed for protein synthesis and nucleotides used as basic molecules for RNA and DNA synthesis (Hirel and Lea, 2001). The GS/GOGAT cycle is the major mechanism of ammonium assimilation in higher plants regardless of the various sources of ammonium listed above. However, it has often been argued that other enzymes have the capacity to assimilate ammonium, leading to the hypothesis that alternative pathways might operate under particular physiological conditions when the GS/GOGAT pathway may not be able to fulfill its function (Harrison et al., 2003).One of these alternative pathways is the reaction catalyzed by the mitochondrial NAD(H)-dependent Glu dehydrogenase (GDH; EC 1.4.1.2), which possesses the ...

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Section: Enzymatic Assay and Determination Of Total Soluble Proteinmentioning

confidence: 99%

Glutamate Dehydrogenase of Tobacco Is Mainly Induced in the Cytosol of Phloem Companion Cells When Ammonia Is Provided Either Externally or Released during Photorespiration

et al. 2004

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“…For native-PAGE and SDS-PAGE, an equal amount of NADH-GDH activity (0.250 unit) was added per lane. To identify different GDH isoenzymes, proteins were separated in a native 5% polyacrylamide gel and stained for NAD-GDH activity as described by Turano et al (1996). Identical gels were incubated in the GDH stain solution minus Glu as negative controls.…”

Section: Cel Electrophoresis Cel-staining Procedures and Lmmunoblotmentioning

confidence: 99%

“…The amination reaction was used routinely to determine activity and the amount of GDH loaded onto gels as described by Turano et al (1996).…”

Section: Protein Determinations and Enzyme Assaysmentioning

confidence: 99%

Characterization and Expression of NAD(H)-Dependent Glutamate Dehydrogenase Genes in Arabidopsis

et al. 1997

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Two distinct cDNA clones encoding NAD(H)-dependent glutamate dehydrogenase (NAD[H]-GDH) in Arabidopsis thaliana were identified and sequenced. The genes corresponding to these cDNA clones were designated GDH1 and GDH2. Analysis of the deduced amino acid sequences suggest that both gene products contain putative mitochondrial transit polypeptides and NAD(H)- and α--ketoglutarate-binding domains. Subcellular fractionation confirmed the mitochondrial location of the NAD(H)-GDH isoenzymes. In addition, a putative EF-hand loop, shown to be associated with Ca2+ binding, was identified in the GDH2 gene product but not in the GDH1 gene product. GDH1 encodes a 43.0-kD polypeptide, designated α-, and GDH2 encodes a 42.5-kD polypeptide, designated [beta]. The two subunits combine in different ratios to form seven NAD(H)-GDH isoenzymes. The slowest-migrating isoenzyme in a native gel, GDH1, is a homohexamer composed of α- subunits, and the fastest-migrating isoenzyme, GDH7, is a homohexamer composed of [beta] subunits. GDH isoenzymes 2 through 6 are heterohexamers composed of different ratios of α- and [beta] subunits. NAD(H)-GDH isoenzyme patterns varied among different plant organs and in leaves of plants irrigated with different nitrogen sources or subjected to darkness for 4 d. Conversely, there were little or no measurable changes in isoenzyme patterns in roots of plants treated with different nitrogen sources. In most instances, changes in isoenzyme patterns were correlated with relative differences in the level of α- and [beta] subunits. Likewise, the relative difference in the level of α- or [beta] subunits was correlated with changes in the level of GDH1 or GDH2 transcript detected in each sample, suggesting that NAD(H)-GDH activity is controlled at least in part at the transcriptional level.

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“…Fd-GOGAT, nitrate reductase (NR) and GS were assayed as described by Gibon et al [24] and Migge et al [25], respectively. We used the method of Scheible et al [26] to assay nitrite reductase (NiR) and that of Turano et al [27] to assay glutamate dehydrogenase (GDH). Malate dehydrogenase (NADP-MDH) was assayed as described by Jenner et al [28] and pyruvate kinase (PK) as described by Borsani et al [29].…”

Section: Enzyme Assaysmentioning

confidence: 99%

Suppression of glutamate synthase genes significantly affects carbon and nitrogen metabolism in rice (Oryza sativa L.)

Luo

Yang

et al. 2011

Sci. China Life Sci.

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Rice (Oryza sativa) glutamate synthase (GOGAT, EC 1.4.1.14) enzymes have been proposed to have great potential for improving nitrogen use efficiency, but their functions in vivo and their effects on carbon and nitrogen metabolism have not been systematically explored. In this research, we analyzed transcriptional profiles of rice GOGAT genes using a genome-wide microarray database, and investigated the effects of suppression of glutamate synthase genes on carbon and nitrogen metabolism using GOGAT co-suppressed rice plants. Transcriptional profiles showed that rice GOGAT genes were expressed differently in various tissues and organs, which suggested that they have different roles in vivo. Compared with the wild-type, tiller number, total shoot dry weight, and yield of GOGAT co-suppressed plants were significantly decreased. Physiological and biochemical studies showed that the contents of nitrate, several kinds of free amino acids, chlorophyll, sugars, sugar phosphates, and pyridine nucleotides were significantly decreased in leaves of GOGAT co-suppressed plants, but the contents of free ammonium, 2-oxoglutarate, and isocitrate in leaves were increased. We conclude that GOGATs play essential roles in carbon and nitrogen metabolism, and that they are indispensable for efficient nitrogen assimilation in rice.glutamate synthase, Oryza sativa, co-suppression, carbon and nitrogen metabolism Citation:Lu Y E, Luo F, Yang M, et al. Suppression of glutamate synthase genes significantly affects carbon and nitrogen metabolism in rice (Oryza sativa

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Purification of Mitochondrial Glutamate Dehydrogenase from Dark-Grown Soybean Seedlings

Cited by 97 publications

References 26 publications

Glutamate Dehydrogenase of Tobacco Is Mainly Induced in the Cytosol of Phloem Companion Cells When Ammonia Is Provided Either Externally or Released during Photorespiration

Glutamate Dehydrogenase of Tobacco Is Mainly Induced in the Cytosol of Phloem Companion Cells When Ammonia Is Provided Either Externally or Released during Photorespiration

Characterization and Expression of NAD(H)-Dependent Glutamate Dehydrogenase Genes in Arabidopsis

Suppression of glutamate synthase genes significantly affects carbon and nitrogen metabolism in rice (Oryza sativa L.)

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