A Plastidial Localization and Origin of l-Glutamate Dehydrogenase in a Soybean Cell Culture

Abstract

The subcellular distribution of L-glutamate dehydrogenase (GDH, EC 1.4.1.3.) was studied in SB3 soybean (Glycine max) cells using subcellular fractionation techniques. Compounds that inhibit protein synthesis either on 80s or 70s ribosomes were also used to give a preliminary idea of which subcellular fraction is involved in GDH synthesis. It was found that whereas cycloheximide and puromycin considerably reduced the total amount of protein synthesized by the cells, they did not appear to inhibit the synthesis of GDH. In the presence of chloramphenicol, both GDH activity and protein level in the cells were considerably reduced, suggesting that this enzyme was synthesized in organelles and not in the cytosol. Streptomycin, which inhibits plastid protein synthesis, also inhibited synthesis of GDH, indicating that a fraction of GDH activity was plastidial in origin. This is supported by the data on subcellular distribution of the enzyme, which showed that a major fraction of GDH is found in the plastidial fraction, although some activity is found associated with the mitochondrial fraction also. Since a major fraction of GDH activity was found in the plastidial fraction, we studied protein synthesis using isolated plastids and 35S-methionine. Using antibodies raised against purified GDH, we identified a 35S-labeled 41-kilodalton polypeptide synthesized by plastids as GDH.

Metabolic Inhibitors, Elicitors, and Precursors as Tools for Probing Yield Limitation in Taxane Production by Taxuschinensis Cell Cultures

“…Since isoprenoid biosynthesis occurs in more than one cellular compartment, it is important to understand the localization of taxane production before addressing yield improvement strategies such as precursor feeding or elicitation. Cycloheximide (CH) and streptomycin (SS) have been used widely as tools to distinguish between cytoplasmic and plastidic mRNA translation events (Bhadula and Shargool, 1991; Mita et al, 1995). Therefore, if taxane production had a significant plastid component, we would expect the process to be sensitive to SS, and if the biosynthesis was predominantly cytoplasmic, we would expect the process to be sensitive to CH.…”

Implication of the glutamine synthetase/glutamate synthase pathway in conditioning the amino acid metabolism in bundle sheath and mesophyll cells of maize leaves

“…By contrast, members of the GDH S‐50 I class, such as plastidial NADPH‐GDH (EC 1.4.1.4) of Chlorella , assimilate ammonium into glutamate [46]. In fact, chloroplast NADPH‐GDH is found in higher plants [47], suggesting a possible alternative role of this isoform. Therefore, NADH‐GDH may provide the anaplerotic pathway with 2‐oxoglutarate to regenerate NADH and 2‐oxalacetate for further transaminations.…”

Modulation of higher-plant NAD(H)-dependent glutamate dehydrogenase activity in transgenic tobacco via alteration of beta subunit levels

“…A protein database search indicated that At1g51720 was most similar to an NADP(H)-dependent GDH (EC 1.4.1.4) from the rodent malaria parasite Plasmodium yoelii yoelii (70% similarity over 438 AA; accession EAA20557), and was also 63% similar (over 437 AA) to an NADP(H)-dependent GDH from the unicellular alga Chlorella sorokiana (accession CAA41636). At1g51720 is transcriptionally active (Yamada et al 2003), and hence vascular plants may also contain a functional NADP(H)-dependent GDH isoform, despite there being hitherto only limited evidence of this possibility (Lea and Thurman 1972;Bhadula and Shargool 1991).…”