Localisation of Glutamine Synthetase in Chloroplasts

203

Density gradient separation of plastids from leaf and root tissue was carried out. The distribution in the gradients of the activity of the following enzymes was determined: nitrite reductase, glutamine synthetase, acetolactate synthetase, aspartate amninotransferase, catalase, cytochrome oxidase, and triosephosphate isomerase. The distribution of chlorophyll was followed in gradients from leaf tissue. The presence of plastids that have retained their stroma enzymes was denoted by a peak of triosephosphate isomerase activitv. Coincidental with this peak were bands of nitrite reductase, acetolactate synthetase, glutamine synthetase, and aspartate aminotransferase activity. The results suggest that most, if not all, the nitrite reductase and acetolactate synthetase activity of the cell is in the plastids. The plastids were found to contain only part of the total glutamine synthetase, aspartate aminotransferase, and triosephosphate dehydrogenase activity in the cell. Some evidence was obtained for low levels of glutamate dehydrogenase activity in chloroplasts.A proportion of the nitrite reductase extracted from plant cells is associated with a particulate fraction in both leaves and roots (10,21). While most work has suggested that nitrite reductase is localized in the chloroplast, Lips and Avissar (17) have suggested that the enzyme is present in the peroxisome. Miflin (21) found that the enzyme from barley roots banded in sucrose density gradients below the mitochondria and microbodies but he did not assay for any plastid markers and was unable to identify the organelle in question. Subsequently, Dalling et al. (7) found that nitrite reductase from wheat roots, under isopycnic density gradient centrifugation, was distributed coincidently with only one of two peaks of plastid marker enzymes. Because of the unusual distribution of the plastid markers in the gradient and the lack of complete coincidence between them and nitrite reductase, it is not possible from these results to state unequivocally that nitrite reductase is located in root plastids.If it is accepted that nitrite reductase is present in the plas- (19,20,23). Leech and co-workers (9, 16) and Lea and Thurman (15) have shown that chloroplasts have a light-stimulated glutamate dehydrogenase, which appears to be NADPH-dependent and bound tightly to the chloroplast lamellae. There is also evidence that chloroplasts can incorporate ammonia into glutamine (12,25,27). Further, chloroplasts contain a wide range of transaminases (13). In root tissues, glutamate dehydrogenase seems to be solely in the mitochondria (7,21), but, apart from these observations, little seems to have been done on the location of these enzymes in roots.Studies on "4CO2 fixation in chloroplasts indicate that "4C does not readily appear in amino acids, which suggests that the carbon skeletons for the amino acids are derived from outside the chloroplasts. In agreement with this a-ketoglutarate, pyruvate, and P-enolpyruvate stimulate the formation of a-amino nitrogen from nitrite...

mentioning

confidence: 99%

The Location of Nitrite Reductase and Other Enzymes Related to Amino Acid Biosynthesis in the Plastids of Root and Leaves

Miflin¹

1974

203

“…Further studies on chloroplastic glutamine synthesis indicated that ATP required for the operation of GS3 was provided by photophosphorylation (1,6,14). GS has also been shown to be present in chloroplasts by means of direct enzyme assays on Pisum (16) and Vicia chloroplasts (11). Chlo total leaf GS (16,29).…”

mentioning

confidence: 99%

“…GS has also been shown to be present in chloroplasts by means of direct enzyme assays on Pisum (16) and Vicia chloroplasts (11). Chlo total leaf GS (16,29). There is evidence that GS plays a major role in nitrogen assimilation by plant tissues and that GS works in conjunction with glutamate synthase to produce glutamate (13,23).…”

mentioning

confidence: 99%

Effects of Light and Inhibitors on Glutamate Metabolism in Leaf Discs of Vicia faba L

Jordan

Givan²

1979

Metabom of ("Ciglutamate was studied in leaf discs of Viciafaba L.in lght and in darkness. In white lht a was the main labeled product. In Santarius and Stocking (20) first demonstrated that illuminated chloroplasts, isolated from Spinacia leaves, could synthesize glutamine from glutamate. Further studies on chloroplastic glutamine synthesis indicated that ATP required for the operation of GS3 was provided by photophosphorylation (1,6,14). GS has also been shown to be present in chloroplasts by means of direct enzyme assays on Pisum (16) Leaf laminae were removed from these plants and discs cut from the tissue using a 1-cm cork borer. For each experimental sample 10 discs were used, which gave a frsh weight of about 0.1 g. Discs were placed in 0.2 cm3 of solution containing 1 ,uCi of radioactive substrate, plus any other compound that was required for the experiment, and subjected to vacuum infiltration. Radioactive substrates entered the discs over the entire area and were not restricted to the cut margins.After infiltration, discs were thoroughly washed in distilled H20 and placed under the appropriate incubation conditions for 2 h (except where different time periods are specified) in a temperature-controlled water bath at 25 C. White light was provided by a bank of warm white fluorescent strip lights giving an irradiance of 14.0 w/m2 at the level of the discs. In experiments involving monochromatic light, quartz-iodine projection lamps were fitted with interference filters to provide the light quality required. The transmission of the far red filter peaked at 711.4 nm and the red filter at 651 nm with bandwidths at 50% transmission of 11.0 and 10.4 nm, respectively. The irradiance of these two wavelengths at the level of the discs was 23 w/m2 and 27 w/m2, respectively. A YSl-Kettering model 65A radiometer was used to determine irradiance values.In experiments where CO2 fixation was measured discs were incubated in a bathing solution containing 10 MCi of sodium ['4Clbicarbonate. Anaerobic conditions, where required, were obtained by flushing with 02-free nitrogen gas. This gassing treatment caused a temperature drop of less than 1 C over the incubation period.

“…The newly discovered enzymes, glutamine synthetase and glutamate synthase, in leaves of C3 plants has led to the proposal of an alternate pathway for nitrate assimilation (16). The enzyme glutamine synthetase catalyzes the ATP-driven addition of ammonia to glutamate (10,21,22). The amide group of glutamine is then transferred to aketoglutarate by glutamate synthase (16).…”

mentioning

confidence: 99%

Distribution of Nitrate-assimilating Enzymes between Mesophyll Protoplasts and Bundle Sheath Cells in Leaves of Three Groups of C₄ Plants

Rathnam

Edwards²

1976

Intercellular distribution of enzymes involved in amino nitrogen synthesis was studied in leaves of species representing three C4 groups, i.e. Sorghum bicolor, Zea mays, Digitaria sanguinalis (NADP malic enzyme type); Panicum miliaceum (NAD techniques for separation of mesophyll and bundle sheath cells and also because of the recent progress in understanding the route of amino acid biosynthesis in leaves of C3 plants. The suggestion that glutamate dehydrogenases are responsible for the incorporation of nitrogen into the a-amino group of amino acids through the reductive amination of a-ketoglutarate in presence of ammonia (3) is now regarded by Lea and Miflin (16) as a minor or insignificant pathway. The newly discovered enzymes, glutamine synthetase and glutamate synthase, in leaves of C3 plants has led to the proposal of an alternate pathway for nitrate assimilation (16). The enzyme glutamine synthetase catalyzes the ATP-driven addition of ammonia to glutamate (10,21,22). The amide group of glutamine is then transferred to aketoglutarate by glutamate synthase (16).In leaves, the biological reduction of nitrate to ammonia and the subsequent assimilation of ammonia to organic form utilize photosynthetic energy (3). Since C4 plants have two photosynthetic cell types, mesophyll and bundle sheath, it is of interest to consider the mechanism and localization of nitrate-assimilating enzymes in these species. We utilized species from the three groups of C4 plants in the present work for studying nitrateassimilating enzyme distribution, and the results are discussed in relation to the glutamine synthetase-glutamate synthase pathway of nitrogen assimilation.In C4 plants, the amino acid aspartate is an early labeled product of photosynthesis apparently associated with the C4 pathway (6, 9, 24) rather than being formed from an intermediate of the carbon reduction pathway as occurs in C3 plants (29). Aspartate synthesis in C4 plants takes place in their mesophyll cells (12), and a continual shuttle of aspartate and alanine is proposed between mesophyll and bundle sheath cells which would maintain a nitrogen and carbon balance between the cell types (13,25).In view of this, there has been recent interest in elucidating the mechanism of a-amino nitrogen synthesis in leaves of C4 plants (19,23 MATERIALS AND METHODS Plant Material. The plants used were Zea mays L., Sorghum bicolor (L.) Moench., Digitaria sanguinalis (L.) Scop., Panicum miliaceum L., and Panicum maximum Jacq. The plants were grown in a growth chamber under a daily regime of 16 hr light and 8 hr dark with a day temperature of 30 C and a night temperature of 20 C. Light was provided by a combination of incandescent and fluorescent lamps giving a quantum flux density of 50 to 70 nanoeinsteins/cm2* sec between wavelengths of 400 and 700 nm. The plants were watered daily with nutrient solution described earlier by Beevers et al. (4). Mature leaves from plants 10 to 20 days old were excised after an initial 1 to 2 hr of light exposure following the 8-hr dark...