Leaf Glutamine Synthetase Research Articles

Glutamine synthetases from rice: Purification and preliminary characterization of two forms in leaves and one form in roots

A relatively rapid five-step procedure was used in purifying to apparent homogeneity the glutamine synthetase from roots and one form of the enzyme (GS I) from leaves of rice. The steps were: preparation of crude extracts, ammonium sulfate precipitation, filtration on Sepharose 4B, fractionation on DEAE-Sephadex A25, and affinity chromatography on ADP-Sepharose 4B. The purified protein appeared as a single band on polyacrylamide gel electrophoresis. Leaf GS I and the second type of leaf glutamine synthetase (GS II) formed distinct peaks when eluted from DEAE-Sephadex (step 4). The root enzyme and leaf GS I were similar in all the properties which were examined. Both enzymes bound to ADP-Sepharose, had similar biosynthetic (18 μmol P/ i mg protein/min) and transferase (1324 and 1156 μmol γ-glutamyl hydroxamate/mg protein/min) activities, and the same or nearly the same K m values for glutamate (2.17 m m), Mg 2+ (4.5 and 5.0 m m), ATP (286 μ m), NH 4 + (210 and 135 μ m), and ADP (3.8 and 5.3 μ m). In contrast, leaf GS II did not bind to ADP-Sepharose and had much higher K m values for glutamate (8.3 m m), Mg 2+ (15 m m), NH 4 + (684 μ m), and ADP (33 μ m).

Activity profiles of enzymes involved in glutamine and glutamate metabolism in the apple during autumnal senescence

AbstractSeasonal changes in glutamine synthetase (EC 6.3.1.2), glutamate synthase (EC 2.6.1.53), and glutamate dehydrogenase (EC 1.4.1.3) were measured in both senescing leaf and bark tissues of ‘Golden Delicious’ apple trees (Malus domestica Borkh.). From the measured enzyme activities we attempted to estimate the in vivo catalytic potentials of the enzymes with special reference to nitrogen mobilization and conservation of senescing apple trees. The cumulative glutamine synthetase activity of leaf tissue was about three times higher than that of bark. The estimated catalytic potential of leaf glutamine synthetase was 800‐fold higher than the actual protein nitrogen loss of senescing leaves. The cumulative glutamate synthase activity of bark was about six times higher than that of leaf. The estimated catalytic potential of bark glutamate synthase was 160‐times higher than the actual protein nitrogen gain in that tissue. The cumulative glutamate dehydrogenase activities in leaf and bark tissue were approximately the same. However, the catalytic potential of leaf glutamate dehydrogenase was twice that of leaf glutamate synthase. It is thus concluded that the physiological role of glutamine synthetase in senescing leaf tissue is to furnish the amide(s) prior to mobilization of nitrogen to storage tissue. The higher activity of glutamate synthase in bark tissue could provide a mechanism to transform the imported amide nitrogen to amino nitrogen of glutamate for storage protein synthesis. The possible regulatory factors upon the activity of these enzymes in the tissues of senescing apple trees are discussed.

Enzymology of Glutamine Metabolism Related to Senescence and Seed Development in the Pea (Pisum sativum L.)

The metabolism of glutamine in the leaf and subtended fruit of the aging pea (Pisum sativum L. cv. Burpeeana) has been studied in relation to changes in the protein, chlorophyll, and free amino acid content of each organ during ontogenesis. Glutamine synthetase [EC 6.3.1.2] activity was measured during development and senescence in each organ. Glutamate synthetase [EC 2.6.1.53] activity was followed in the pod and cotyledon during development and maturation. Maximal glutamine synthetase activity and free amino acid accumulation occurred together in the young leaf. Glutamine synthetase (in vitro) in leaf extracts greatly exceeded the requirement (in vivo) for reduced N in the organ. Glutamine synthetase activity, although declining in the senescing leaf, was sufficient (in vitro) to produce glutamine from all of the N released during protein hydrolysis (in vivo). Maximal glutamine synthetase activity in the pod was recorded 6 days after the peak accumulation of the free amino acids in this organ.In the young pod, free amino acids accumulated as glutamate synthetase activity increased. Maximal pod glutamate synthetase activity occurred simultaneously with maximal leaf glutamine synthetase activity, but 6 days prior to the corresponding maximum of glutamine synthetase in the pod. Cotyledonary glutamate synthetase activity increased during the assimilatory phase of embryo growth which coincided with the loss of protein and free amino acids from the leaf and pod; maximal activity was recorded simultaneously with maximal pod glutamine synthetase.We suggest that the activity of glutamine synthetase in the supply organs (leaf, pod) furnishes the translocated amide necessary for the N nutrition of the cotyledon. The subsequent activity of glutamate synthetase could provide a mechanism for the transfer of imported amide N to alpha amino N subsequently used in protein synthesis. In vitro measurements of enzyme activity indicate there was sufficient catalytic potential in vivo to accomplish these proposed roles.

Pea Leaf Glutamine Synthetase

Of a variety of purine and pyrimidine nucleotides tested, only ADP and 5'AMP significantly inhibited the Mg(2+)-dependent activity of pea leaf glutamine synthetase. They were less effective inhibitors where Mn(2+) replaced Mg(2+). They were competitive inhibitors with respect to ATP, with inhibition constant (Ki) values of 1.2 and 1.8 mm, respectively. The energy charge significantly affects the activity of glutamine synthetase, especially with Mg(2+). Of a variety of amino acids tested, l-histidine and l-ornithine were the most inhibitory, but significant inhibition was seen only where Mn(2+) was present. Both amino acids appeared to compete with l-glutamate, and the Ki values were 1.9 mm for l-histidine (pH 6.2) and 7.8 mm for l-ornithine (pH 6.2). l-Alanine, glycine, and l-serine caused slight inhibition (Mn(2+)-dependent activity) and were not competitive with ATP or l-glutamate.Carbamyl phosphate was an effective inhibitor only when Mn(2+) was present, and did not compete with substrates. Inorganic phosphate and pyrophosphate caused significant inhibition of the Mg(2+)-dependent activity.