A PLASTIDIAL LOCALIZATION AND ORIGIN OF L-GLUTAMATE DEHYDROGENASE IN A SOYBEAN CELL-CULTURE

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 S-35-methionine. Using antibodies raised against purified GDH, we identified a S-35-labeled 41-kilodalton polypeptide synthesized by plastids as GDH.