Phosphorylation of methionine sulfoximine by glutamine synthetase.

Ronzio, Robert A.; Meister, Alton

doi:10.1073/pnas.59.1.164

Cited by 156 publications

(55 citation statements)

References 20 publications

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“…Monochromatic light (710 nm) in the presence of DCMU, caused a 2-fold increase in glutamine accumulation, compared to the dark control. The ratio of labeled malate to aspartate, while it could not be meaningfully compared with the ratio in darkness under anaerobic conditions (since both values were zero), was substantially greater than that seen in the dark under aerobic conditions (Table I) MSO, an inhibitor of GS (19), was employed in order to see how glutamate would be metabolized in the light in the absence of glutamine formation (Table VI) (10,27). If the synthesis of glutamine is inhibited in the light, label from glutamate rapidly enters intermediates of the tricarboxylic acid cycle, which are to a large degree mitochondrial in location.…”

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confidence: 99%

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

Jordan

Givan²

1979

Plant Physiol.

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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.

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confidence: 99%

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

Jordan

Givan²

1979

Plant Physiol.

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“…Finally, GOGAT activity in Caulerpa chloroplasts, like the enzyme in plants (15) and bacteria (4), was inhibited by azaserine (Table II), but not by methionine sulfoximine, a potent inhibitor of glutamine synthetase (16). Glutamine Synthetase Activity in C. simpliciusculs Chloroplasts.…”

Section: Resultsmentioning

confidence: 99%

Glutamine Synthetase/Glutamine: α-Ketoglutarate Aminotransferase in Chloroplasts from the Marine Alga Caulerpa simpliciuscula

McKenzie¹,

Ch'Ng²,

Gayler³

1979

Plant Physiol.

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The enzymic capacities for ammonia assimilation into amino acids have been investigated in chloroplasts from the siphonous green alga Caulerpa simpliciuscula (Turner) C. Ag. The results show that these chloroplasts differ from those of higher plants in having present simultaneously the enzymic capacities to permit assimilation of ammonia by two pathways.Glutamine synthetase (EC 6.3.1.2) activity at levels up to 4 jumoles per mg chlorophyll per hour were found in soluble extracts of the chloroplasts. Glutamine(amide):a-ketoglutarate aminotransferase (oxidoreductase ferredoxin) (EC 1.4.7.1) activity at levels up to 1.4 pmoles per mg chlorophyll per hour was detected by incubation of photosynthetically active chloroplasts either in light or with reduced ferredoxin. Together these enzymes provide the capacity for the conventional pathway of ammonium assimilation in chloroplasts via glutamine. A similar level of a glutamate dehydrogenase with an unusually low Km for ammonia which has been described previously in these chloroplasts provides the second potential pathway.Chloroplasts isolated from siphonous green algae have been shown recently to synthesize a different, and often wider, array of photosynthetic products from ''CO2 than higher plant chloroplasts (7,19). In this they resemble the chloroplasts isolated from Acetabularia where a similar range of products is synthesized by the isolated organelle (17). In this paper the studies of the metabolic competence of chloroplasts from siphonous green algae are extended to include the pathways of assimilation of ammonium into amino acids.In chloroplasts of higher plants ammonium is incorporated into amino acids via the two enzymic steps, glutamine synthetase (EC 6.3.1.2) followed by GOGAT2 (Jo). It has been suggested that the situation in chloroplasts isolated from the marine alga Caulerpa simpliciuscula is different, since they have been found to contain high levels of an NADP-dependent glutamate dehydrogenase, with an abnormally low apparent Km for ammonia of 0.4 to 0.7 mM (5). This enzyme could provide an alternative means of ammonium incorporation into amino acids in these algae.GOGAT activity has been demonstrated both in photosynthetic organisms which are more advanced in evolution than C. simpliciuscula and also in those which are more primitive (10, 11). We would expect that GOGAT activity should also be present in C. simpliciuscula unless this species represents a radical departure in evolution from other members of the plant kingdom. Here, we show that the chloroplasts of this siphonous green alga contain the enzymic capacities for both pathways of ammonium assimilation into amino acids. MATERIALS AND METHODSPlant Material. C. simpliciuscula plants were gathered at a depth of 1 to 2 m from the sea at Williamstown, Victoria and held in an illuminated tank of aerated and recirculating seawater on a 12-h light/12-h dark cycle at 16 C.Chloroplast Isolation. The chloroplast fraction was prepared from fronds of C. simpliciuscula by the method described prev...

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“…Y-to-M transition studies were also carried out with inhibitors such as cycloheximide (a protein synthesis inhibitor), isophthalic acid (a strong competitive NAD-GDH inhibitor) (14), methionine sulfoximine (an irreversible GS inhibitor) (11), and azaserine, a glutamine analog (a GOGAT inhibitor) (4).…”

Section: Resultsmentioning

confidence: 99%

Significance of NADP/NAD glutamate dehydrogenase ratio in the dimorphic behavior of Benjaminiella poitrasii and its morphological mutants

1992

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Studies on the levels of glutamate dehydrogenase (GDH), glutamine synthetase, and glutamate synthase were carried out as a function of temperature, nutritional conditions, and the morphological (yeast or mycelium) form of Benjaminiella poitrasii. Since both NAD-and NADP-dependent GDH activities were found in B. poitrasii, the quantitative relation between these two enzymes expressed as the NADP-GDH/NAD-GDH activity ratio (GDH ratio) was studied to evaluate its possible role in the morphogenesis. In the yeast-to-mycelium transition, a decrease in the GDH ratio occurred (between 1 and 2 h) and germ tube formation could be observed only at 3 h. Under similar sets of experimental conditions, exogenous addition 1.0 mM of a-ketoglutarate delayed germ tube emergence (4 h) compared with the control. On the other hand, in the presence of 1.0 mM glutamate an earlier onset of the germ tube formation was noted. The morphological (monomorphic) mutants, Y-2 and Y-5, showed a high GDH ratio and maintained the yeast morphology.The dimorphic zygomycete Benjaminiella poitrasii has the potential to differentiate into alternative morphologies depending on growth temperature and/or nutritional conditions. The organism grows in mycelial form (M form) at 28°C whereas development of the yeast form (Y form) occurs at 37°C. In addition, optimal yeast growth requires the presence of a hexose as a carbon source as well as an organic amino compound as a nitrogen source. In contrast, the mycelial growth occurs on a variety of carbon sources and with ammonium salts as the sole nitrogen source (5). These observations suggest differential expression of some key enzymes of carbon and nitrogen metabolism in the two morphological forms. Generally, in fungi, the carbon and nitrogen metabolisms are connected via ammonia assimilation and the participating enzymes are NAD-dependent (EC 1.4.1.2) and NADP-dependent (EC 1.4.1.4) glutamate dehydrogenase (GDH), glutamate synthase (GOGAT; EC 1.4.1.13), and glutamine synthetase (GS; EC 6.3.1.2) (8).Since the dimorphic (Y-to-M) transitions in B. poitrasii can be regulated by the carbon-nitrogen balance, the changes in the levels of the aforementioned enzymes were studied to evaluate their possible role. Similar studies were also carried out with monomorphic Y form mutants (Y-2 and Y-5) isolated after N-methyl-N'-nitro-N-nitrosoguanidine treatment of parent strain spores (5). (pH 7.2) was prepared. The pH was adjusted to 7.2 with 2 M NaOH or 1.0 M HCI. The reaction mixture (1 ml) containing 0.4 ml of the above assay mixture, 0.2 ml of crude extract, and 0.35 ml of glass-distilled water was preincubated for 5 min at 37°C. Then the reaction was initiated by the addition MATERUILS AND METHODS

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Phosphorylation of methionine sulfoximine by glutamine synthetase.

Cited by 156 publications

References 20 publications

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

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

Glutamine Synthetase/Glutamine: α-Ketoglutarate Aminotransferase in Chloroplasts from the Marine Alga Caulerpa simpliciuscula

Significance of NADP/NAD glutamate dehydrogenase ratio in the dimorphic behavior of Benjaminiella poitrasii and its morphological mutants

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