Characterization of glutamine‐synthetase from Beta vulgaris

Neßelhut, Thomas; Harnischfeger, G.

doi:10.1111/j.1399-3054.1981.tb05563.x

Cited by 12 publications

(7 citation statements)

References 19 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Leaf segments were homogenized under ice‐cold conditions with a mortar and pestle in the presence of some acid‐washed sea sand. For the assay of glutamine synthetase the homogenates were prepared according to Nesselhut & Harnischfeger (1981) in 50 mol m −3 imidazole buffer (pH 7·5), containing 15 mol m −3 MgSO 4 , 8 mol m −3 2‐mercaptoethanol and 10 mol m −3 ATP. For the assay of the other enzyme activities, extracts were prepared in the presence of 50 mol m −3 K‐phosphate buffer, pH 7·5, as described ( Streb et al 1997 ).…”

Section: Methodsmentioning

confidence: 99%

“…Enzyme activities for catalase (EC 1·11·1.6), glycolate oxidase (EC 1·1.3·1), glutathione reductase (EC 1·6.4·2), guaiacol‐dependent peroxidase (EC 1·11·1.7), and superoxide dismutase (EC 1·15·1.1) were assayed spectrophotometrically at 25 °C, as described previously ( Streb et al 1997 ). Glutamine synthetase (EC 6·3.1·2) activity was assayed as the hydroxylamine‐dependent synthetase reaction according to Nesselhut & Harnischfeger (1981).…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Significance of antioxidants and electron sinks for the cold‐hardening‐induced resistance of winter rye leaves to photo‐oxidative stress

Streb

Feierabend

1999

Plant Cell & Environment

View full text Add to dashboard Cite

The contents of ascorbate and glutathione and the activities of superoxide dismutase and glutathione reductase were increased to levels as high as those in cold-hardened leaves (CHL) by incubating non-hardened leaves (NHL) of winter rye (Secale cereale L.) with the precursor substrates L-galactonic acid-g g-lactone and 2-oxothiazolidine-4-carboxylate. Reduced glutathione was rapidly depleted from NHL after application of D,L-buthionine sulfoximine, an inhibitor of its biosynthesis. In spite of greatly divergent antioxidant contents the rates of photo-inactivation of photosystem II (PSII) and catalase observed in the presence of translation inhibitors did not differ greatly. The paraquatinduced catalase inactivation and chlorophyll degradation in light were reduced in NHL with increased antioxidant levels. Paraquat-induced photo-inactivation of PSII was, however, not mitigated. The CHL had a higher capacity to prevent paraquat-induced oxidation of ascorbate and glutathione than NHL with increased antioxidant contents. Increased antioxidant contents did not establish resistance to low temperature-induced photo-inactivation of PSII and catalase in NHL. The resistance of CHL to low temperature-induced photo-inactivation of PSII and catalase required repair at low temperature and active carbon assimilation but was only little affected when photorespiration was suppressed by phosphinothricin. Protection of PSII depended also on non-photochemical quenching of excitation energy.

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Significance of antioxidants and electron sinks for the cold‐hardening‐induced resistance of winter rye leaves to photo‐oxidative stress

Streb

Feierabend

1999

Plant Cell & Environment

View full text Add to dashboard Cite

show abstract

“…GS activity was determined using the hydroxylaminedependent synthetase assay as described by Nesselhut and Harnischfeger (1981). Foliowing a 30 min incubation period at 25°C in 40 mM Giu, 20 mM MgCl,, 10 mM freshly prepared HONH,C1 and 5 mM ATP, the enzymatic reation was terminated by the addition of 1,5 ml of a soiution containing 0.37 M FeCl,, 0.2 M TCA and 0.67 M HCi and the amount of Fe-glutamylhydroxamate determined at 535 nm, Fd-GOGAT, asparaginase, giutaminase and asparagine aminotransferase activities were determined by incubation of the assay medium at 25°C for 30 min and stopping the enzymatic reaction by the addition of 0.25 ml 15% (w/v) TCA.…”

Section: Enzyme Assaysmentioning

confidence: 99%

Ammonium and amino acid metabolism in excised leaves of wheat (Triticum aestivum) senescing in the dark

Peeters

Laere

1992

Physiologia Plantarum

View full text Add to dashboard Cite

Several parameters of amino acid metabolism were studied in detached primary leaves of wheat (Triticum aestivum L. cv. Castell) during a 14 day incubation period in the dark. Protein loss was accompanied by a 5‐fold increase in the total amount of free amino acids during the first 4 days of the incubation period with asparagine being the most important. Beyond this stage a pronounced intracellular accumulation of ammonium occured. A gradual decrease in the levels of free amino acids and ammonium at the later stages of senescence could in part be accounted for by leakage from the leaves. Additionally, some nitrogen was lost due to ammonia volatilization. The rapid decay of the glutamine synthetase (GS; EC 6.3.1.2)‐glutamate synthase (Fd‐GOGAT; EC 1.4.7.1) system and the fast decline of glutamate‐pyruvate transaminase (GPT; EC 2.6.1.2) activity appear to be predominant features of senescence in the dark. Decreasing Fd‐GOGAT activity was slightly compensated by a small and temporary increase in the activity of NADH‐GOGAT (EC 1.4.1.14). Glutamateoxalocetate transaminase (GOT: EC 2.6.1.1) activity, although declining continuously, proved to be much more persistent. Changes in glutamate dehydrogenase (GDH; EC 1.4.1.3) activity closely resembled the profile of ammonium evolution in the leaves and NADP‐isocitrate dehydrogenase (IDH; EC 1.1.1.42) activity revealed a temporary maximum during the period of rapid increase in GDH activity. Increased activity of GDH could also be induced by exogenous ammonium. Ammonium accumulation could, at least partly, be caused by increased asparaginase (EC 3.5.1.1) activity which accompanied the rapid conversion of asparagine to aspartic acid. Asparagine aminotransferase (EC 2.6.1.14) activity declined sharply from the beginning of the senescence period. Although the activity profile of glutaminase (EC 3.5.1.2) was similar to that of asparaginase, glutamine was of little importance quantitatively and an analogous relationship between glutamine and glutamic acid could not be detected.

show abstract

“…In the absence of ADP, 27% activity was observed. Glutamine synthetase of Anabaena cylindrica (Sawhncy & Nicholas, 1978) and Beta vulgaris seedlings (Nesselhut & Harnischfeger, 1981) showed 65% and 45% of the activity in the complete assay mixture, respectively, in the absence of ADP. This activity could be due to ADP tightly bound to enzyme molecule.…”

Section: Resultsmentioning

confidence: 99%

Purification and characterization of glutamine synthetase from Clostridium pasteurianum

Krishnan¹,

Singhal²,

Dua³

1986

Biochemistry

View full text Add to dashboard Cite

Glutamine synthetase from Clostridium pasteurianum grown on molasses as the sole carbon source and ammonium chloride as the nitrogen source has been purified to homogeneity (45-fold) with 32% recovery. The procedure involves ammonium sulfate precipitation and chromatography on a combined Sepharose 4B/DEAE-Sephadex A-50 column. The purified enzyme being very unstable was stabilized by the addition of 25% (v/v) glycerol. The enzyme has an unusually high molecular weight of 1 X 10(6) and 20 subunits of Mr 50 000 each, as determined by gel filtration and sodium dodecyl sulfate gel electrophoresis, respectively. It has an absorption maximum at 280 nm and a fluorescence emission maximum at 380 nm when excited at 280 nm. Its substrate binding pattern as studied by fluorescence quenching studies is different from that of the Escherichia coli enzyme. Both the gamma-glutamyltransferase and synthetase activities reside in the same protein as the ratio of the two activities at each step of purification remains constant and the enzyme exhibits optimal transferase and synthetase activities at the same pH (7.2) and temperature (50 degrees C). The thermal stabilities of both activities were also similar, and decay of both the activities at 50 degrees C ran parallel. The enzyme shows stabilization by substrates, as L-glutamate, Mg2+, and ATP + Mg2+ protected both the synthetase and gamma-glutamyltransferase activities against thermal inactivation. Storage in 25% (v/v) glycerol enhanced the thermal stability of glutamine synthetase. Metal ion requirement and substrate specificity of the enzyme have been examined. Maximum synthetase activity occurs when [Mg2+]: [ATP] = 2. The Km app values are as follows (in parentheses): ATP (0.34 mM), NH2OH (0.4 mM in the synthetase reaction and 4.1 mM in the transferase reaction), glutamine (14.7 mM), ADP (3.8 X 10(-4) mM), arsenate (2.5 mM), and L-glutamate (3.4 mM, 22.2 mM). The enzyme exhibits negative cooperativity in the binding of glutamate. Amino acids such as L-serine, glycine, L-alanine, and L-aspartic acid inhibit the enzyme.

show abstract

Characterization of glutamine‐synthetase from Beta vulgaris

Cited by 12 publications

References 19 publications

Significance of antioxidants and electron sinks for the cold‐hardening‐induced resistance of winter rye leaves to photo‐oxidative stress

Significance of antioxidants and electron sinks for the cold‐hardening‐induced resistance of winter rye leaves to photo‐oxidative stress

Ammonium and amino acid metabolism in excised leaves of wheat (Triticum aestivum) senescing in the dark

Purification and characterization of glutamine synthetase from Clostridium pasteurianum

Contact Info

Product

Resources

About