Studies on the Enzymatic Synthesis of Glutamine

Varner, Joseph E.; Webster, George C.

doi:10.1104/pp.30.5.393

Cited by 63 publications

(25 citation statements)

References 18 publications

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“…We conclude that ammonia and glutamine compete for the reaction product which is coupled to 02 evolution (i.e. photosynthetically coupled 25 glutamate synthase).…”

Section: Ammonia Assimilation By Chloroplastsmentioning

confidence: 88%

“…Some characteristics of the (ammonia plus a-ketoglutarate)-dependent 02 evolution from our preparations cannot be interpreted simply on the basis of reactions I to V: the requirement for ADP and Pi, compounds which inhibit glutamine synthetase from pea leaves (21,25); added glutamate had no effect although it inhibited glutamate synthase (1, 17); long preincubation periods were needed to eliminate a lag before ammonia would initiate 02 evolution at maximum rate (Fig. 2); a corresponding lag occurred when a-ketoglutarate was used to initiate the reaction which could not be eliminated by preincubation nor by addition of glutamate.…”

Section: Ammonia Assimilation By Chloroplastsmentioning

confidence: 99%

“…The requirements for ADP and PPi, which inhibit plant glutamine synthetase (21,25), and for MgCl2 are not fully understood. We suspect that they are required for reaction I (4, l) 22) and that a favorable concentration of MgCl2 is also required for reaction 11 (20).…”

Section: Ammonia Assimilation By Chloroplastsmentioning

confidence: 99%

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Polarographic Study of Ammonia Assimilation by Isolated Chloroplasts

Anderson¹,

Done²

1977

Plant Physiol.

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Illuminated pea (Pisum sativum) chloroplasts catalyze (ammonia plus a-ketoglutarate [a-KGJ)-dependent 02 evolution at rates which are commensurate with other estimates of the flux of assimilated nitrogen (mean of eight determinations, 8.3 ,umole per mg chlorophyl per hour, SD 2.4). The reaction was usually initiated with 1 mm ammonia after preincubating chloroplasts in the presence of a-KG, ADP, pyrophosphate, and MgCl2.Progressive increases in ammonia concentration gave Vmax/2 at 0.2 mm (approximately) and Vmax at about 1 mm. Higher concentrations were inhibitory; at 7 mm the rate was again about Vmax/2. The highest ratio of 02 evolved per mol of ammonia supplied was 0.36.The (ammonia plus a-KG)-dependent reaction was inhibited by methionine sulfoximine, azaserine, and aspartate in the presence of amino-oxyacetate but not by amino-oxyacetate alone and not by Lglutamate. The rate of 02 evolution in the presence of 1 mm ammonia and 2.5 mM a-KG was increased only slightly by addition of 5 mM glutamine. Similarly, the rate of 02 evolution in the presence of 5 mM glutamine and 2.5 mM a-KG was increased only slightly by addition of 1 mm ammonia.The results are attributed to the incorporation of ammonia via glutamine synthetase and reductive transamination of the glutamine formed by photosynthetically coupled glutamate synthase using a-KG as the amino acceptor. Several lines of evidence rule out the possibility that photosynthetically coupled glutamate dehydrogenase is involved.It has long been known that glutamate is an early product of ammonia assimilation in plants (3,5). It now seems to be generally agreed that chloroplasts are the main site of reduction of nitrite to ammonia in leaf tissue (13,15). Glutamate dehydrogenase (NADPH-specific), glutamine synthetase, and glutamate synthase have been reported in chloroplasts suggesting that these organelles might also be an important site of ammonia assimilation.Illuminated chloroplasts catalyze the formation of glutamate from a-ketoglutarate (7) in the absence of exogenous ammonia. Ammonia (10 mM) had no significant effect on the reaction but the effect of lower concentrations was not reported. was attributed to photosynthetically coupled4 glutamate dehydrogenase (NADPH-specific). This enzyme is bound to the lamellae of Vicia faba chloroplasts (11). Glutamate dehydrogenase might account for ammonia assimilation in chloroplasts although the rate of glutamate formation (0.44 ,umol mg Chl-V hr-1) is low in comparison with the nitrite assimilation rates of chloroplasts of 6 to 12 ,umol mg Chl-' hr-1 (13) and 8 to 9,umol mg Ch-' hr-' (15). Further, the enzyme from Lactuca sativa chloroplasts has a low affinity for ammonia (10). Mitchell and Stocking (18) have recently shown that illuminated pea chloroplasts catalyze the formation of ['4C]glutamine from ['4C]glutamate in the presence of 1 mm ammonia at initial rates of 21 to 26 ,umol mg Chl-I hr-1. This suggests that glutamine synthetase, an enzyme known to be associated with chloroplasts (19, 23) and which has a hig...

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“…We conclude that ammonia and glutamine compete for the reaction product which is coupled to 02 evolution (i.e. photosynthetically coupled 25 glutamate synthase).…”

Section: Ammonia Assimilation By Chloroplastsmentioning

confidence: 88%

Section: Ammonia Assimilation By Chloroplastsmentioning

confidence: 99%

See 1 more Smart Citation

Polarographic Study of Ammonia Assimilation by Isolated Chloroplasts

Anderson¹,

Done²

1977

Plant Physiol.

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“…(7,19,20), and chick embryos (14). A glutamine synthetase which synthesizes glutamine from glutamic acid and ammonia, or glutamyl hydroxamic acid from glutamic acid and hydroxylamine, with adenosine triphosphate (ATP) as energy donor, has also been reported from various sources, including higher plants (5 synthetase preparations from pea seeds have been shown to have transferase activity even after purification of 1000-to 4000-fold (6,18). Levintow and coworkers (9) subjected a highly purified synthetase preparation from pea seed to ultracentrifugal analysis and showed that both synthetase and transferase activities were associated with a single monodisperse protein component.…”

Section: Livingston G E Anid Markakis P Biosynthesismentioning

confidence: 99%

Amide Metabolism in Higher Plants. III. Distribution of Glutamyl Tranferase and Glutamine Synthetase Activity.

Loomis

1959

Plant Physiol.

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“…With respect to higher plants, the pea seed enzyme has been purified and partially characterized (23,25), as have the carrot (3) and rice (9) enzymes. An earlier paper (16) described the preparation of purified glutamine synthetase from pea leaves, which is perhaps distinct from the pea seed enzyme.…”

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confidence: 99%

Glutamine Synthetase of Pea Leaves

O'Neal

Joy²

1974

Plant Physiol.

104

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(8, 13, 19. 20, 26, 27). The Bacillus subtilis, rat liver, and ovine glutamine synthetases have been similarly investigated (4-6, 14, 18, 21) although not as intensively.With respect to higher plants, the pea seed enzyme has been purified and partially characterized (23,25), as have the carrot (3) and rice (9) enzymes. An earlier paper (16) described the preparation of purified glutamine synthetase from pea leaves, which is perhaps distinct from the pea seed enzyme. In this paper, further information concerning its kinetic properties are reported. MATERIALS AND METHODSThe methods were the same as described in our earlier paper (16). However, in the present study an additional assay method was employed; namely, the transfer assay. In this assay the ability of the enzyme to catalyze the following reaction was measured:Pi, or AsO4 y-glutamyl hydroxamate +NH3 ,y-Glutamyl hydroxamate was determined colorimetrically by the procedure described previously (16). The components of the reaction mixture are given in Table VI.In all cases, the enzyme used in the present studies was at least 75% pure and usually homogenous as determined by disc gel electrophoresis. The enzyme preparations were stored for periods of up to 3 months. RESULTSExcept where noted, the results below were similar using either the modified Pi assay (16) or the glutamyl hydroxamate assay (16).Metal Ion Specificity. The purified enzyme had an absolute requirement for a divalent cation, and Mg`gave the optimal activity. Mn2+ and Co2+ were 45 to 60% and 30 to 45% as effective, respectively, but with Co2' and especially Mn'+, the ratio of concentration of divalent cation and ATP was critical; this ratio strongly influenced the pH optimum (16). Zinc and Fe2+ gave less than 5% of maximal activity, although no studies were made to determine condition (such as pH optimum) for optimal activity with these cations. (Fig. 1), when assayed at pH 7.8. At pH 6.2, however, the addition of 0.5 mM Mn'2 stimulated the reaction by at least 100% (ATP, 8 mm, MgSO4, 20 mM), but only at lower levels (10 mM) of L-glutamate. With 30 mm glutamate, the addition of 0.25 to 1.0 mm Mn2+ caused some inhibition, but much less than that which occurred at pH 7.8 under the same conditions. Part of the inhibition caused by Mn2+ at pH 7.8 is due to a change in pH optimum, from 7.8 to 8.0 (Mg2+ alone) to below 5.8 (Mg2+ plus Mn2+) (Fig. 2). This is similar to the optimum pH for Mn2+ alone (where [Mn'+] = [ATP]) which is about 5.2 (16).

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Studies on the Enzymatic Synthesis of Glutamine

Cited by 63 publications

References 18 publications

Polarographic Study of Ammonia Assimilation by Isolated Chloroplasts

Polarographic Study of Ammonia Assimilation by Isolated Chloroplasts

Amide Metabolism in Higher Plants. III. Distribution of Glutamyl Tranferase and Glutamine Synthetase Activity.

Glutamine Synthetase of Pea Leaves

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