Chloroplastic Glutamine Synthetase Research Articles

Purification and Properties of Glutamine Synthetase from Spinach Leaves

The chloroplastic glutamine synthetase of spinach leaves has been purified to homogeneity using affinity chromatography. This involves a tandem ;reactive blue A-agarose' and ;reactive red-A-agarose' as the final step in the procedure. This procedure results in a yield of 18 milligrams of pure glutamine synthetase per kilogram of starting material. The purity of our enzyme has been demonstrated on both one- and two-dimensional polyacrylamide gels.Purified glutamine synthetase has a molecular weight of 360,000 daltons and consists of eight 44,000 dalton subunits. The K(m) is 6.7 millimolar for glutamate, 1.8 millimolar for ATP (synthetase assay), and 37.6 millimolar for glutamine (transferase assay). The isoelectric point is 6.5 and the pH optima are 7.3 in the synthetase assay and 6.4 in the transferase assay. The irreversible, competitive inhibitors methionine sulfoxamine and phosphinothricin have K(i) values of 0.1 millimolar and 6.1 micromolar, respectively. Amino acid analysis has been carried out and the results compared with published analyses for other isoforms of glutamine synthetase.

The Activity and Isoform Complement of Glutamine Synthetase in Panicum Species Differing in Photosynthetic Pathway

Anion exchange chromatography and immunoprecipitation have been used to demonstrate the presence of two forms (GS 1, and GS 2) of glutamine synthetase in the leaves of nine species of Panicum representative of C 3, C 4 and C 3-C 4 intermediate-type photosynthesis. GS 2 from the Panicum species, P. miliaceum and P. maximum was more thermostable than GS 1, GS 1, and GS 2 from P. laxum were equally thermostable but GS 2 from all the Panicum species examined was more sensitive to inhibition by N-ethylmaleimide than GS 1. GS 1, and GS 2 were characterised as being cytoplasmic and chloroplastic isoforms respectively by their reaction with N-ethylmaleimide and by immunoprecipitation with antibodies raised against the cytosolic isoform in barley and the chloroplastic form in tobacco. C 3 species were found to have higher activity of the chloroplastic isoform of glutamine synthetase than C 4 species. C 3-C 4 intermediate species had total leaf glutamine synthetase activities similar to those in C 3 species but were found to have a lower chloroplastic isoform content. The results are consistent with the reassimilation of photorespiratory ammonia by chloroplastic glutamine synthetase.

The Effect of Thiol Compounds on the Glutamate Affinity of the Chloroplastic Glutamine Synthetase from Mustard Leaves

The chloroplastic glutamine synthetase taken from mustard leaves shows a positive cooperative glutamate binding. The S0.5-value depends on the DTE concentration in the homogenization buffer. Normal glutamate satura­tion kinetics have been found with the root enzyme and the enzyme of etiolated cotyledons. These are not influ­enced by thiol compounds. The possible function of the allosteric behaviour of GS2 is discussed.

Glutamine synthetases of pea leaf and seed cytosol. Structure and properties

Methods for purifying to homogeneity glutamine synthetases ( l-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2) of pea seed cytosol (GS cs) and leaf cytosol (GS cl) have been developed. The gel filtration method was used to estimate GS cs molecular weight as 384 000 and that of GS cl, 520 000. Under electrophoresis in polyacrylamide gel in the presence of SDS, GS cs and GS cl dissociated into monomers of molecular weight 48 000 and 64 000, respectively. The quaternary structures of GS cs and GS cl were investigated by electron microscopy. The two enzymes were found to consist of eight identical monomers each arranged with dihedral point group symmetry 422 ( D 4) at the vertices of two squares. These squares are twisted about the 4-fold axis relative to each other. Unlike GS cl; GS cs is characterized by annular projection of particles in micrographs due to filling of the interior portion of the protein molecule with a stain. GS cs and GS cl secondary structures, calculated on the basis of the circular dichoroism spectra, also differed. The nature of the effects of substrates, M 2+, and methionine sulphoximine on GS cs secondary structure was investigated. The isoelectric point of GS cs was situated at pH 5.15, and that of GS cl at pH 4.8. The N-terminal amino acid of GS cl was alanine, and that of GS cs, glycine. The kinetic characteristics of GS cl and GS cs also differ. GS cs and GS cl, and also leaf chloroplast glutamine synthetase and root glutamine synthetase of pea plant were compared by means of rabbit antibodies against GS cs. The conclusion was made that the multiple molecular forms of glutamine synthetase of pea plant may be isoenzymes.

Chloroplastic glutamine synthetase from tobacco leaves: a glycosylated protein

The amino acid and carbohydrate content of chloroplastic glutamine synthetase from tobacco leaves has been analysed. The enzyme subunit contanins 5% carbohydrate, mainly represented by glucosamine, galactosamine, glucose, galactose and mannose residues. The enzyme subunit displayed a single band of molecular mass 44000 Da after sodium dodecyl sulphate (SDS) electrophoresis. However, when isoelectrofocussing electrophoresis was performed, four subunits were evident differing by their charge. Furthermore, the four different subunits stained positively when tested with periodic acid Shiff reagent, showing that sugars and amino sugars were present within all the subunits.

Glutamine synthetase in ribulose 1,5-bisphosphate carboxylase/oxygenase deficient tobacco mutants in cell suspension culture

In two tobacco mutants lacking ribulose, 1,5-bisphosphate carboxylase/oxygenase the amount of glutamine synthetase and its activity were determined and compared with the wild type green cells. It was shown that in these two mutants glutamine synthetase protein content was six times lower than in the wild type. This situation was comparable to that found in etiolated cells where ribulose 1,5-bisphosphate carboxylase/oxygenase was absent. These observations suggest that a common regulatory mechanism might control the dual light dependent biosynthesis of both enzymes. The results have also implications concerning the efficiency of the reassimilation of ammonia by chloroplastic glutamine synthetase during the photorespiratory process.

Multiple Subunit Composition of Chloroplastic Glutamine Synthetase of Nicotiana tabacum L

Chloroplastic glutamine synthetase from tobacco leaves (Nicotiana tabacum L. var Xanthi) was purified to homogeneity. By using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high performance liquid chromatography, a single subunit was identified with a molecular weight of 45,000 daltons. However the native protein seems to be composed of four different subunits which can be separated by isoelectrofocusing. It is suggested that different genes with eventual posttranslational and/or posttranscriptional modifications may control the synthesis of the chloroplastic glutamine synthetase.

Isoforms of glutamine synthetase in Panicum species having C3, C4, and intermediate photosynthetic pathways

The number, relative activity, and immunological nature of glutamine synthetase (GS) isoforms were studied in three species of Panicum, each differing in the nature of their photosynthetic system: P. bisulcatum, a C3 plant; P. milioides, a C3–C4 intermediate; and P. miliaceum, a C4 plant. Ion-exchange chromatography of leaf extracts for all three species revealed a peak of GS activity eluting at 0.23–0.32 M NaCl. These peaks had similar elution characteristics to GS isolated from chloroplasts of barley. Also, as in the barley chloroplastic GS, they were poorly recognized by antibodies from barley cytosolic GS (GS1). These peaks were therefore identified as GS2 and were thought to be chloroplastic in origin. A second peak of GS activity eluting at 0.18 M NaCl was found to account for 0, 21, and 45% of the total GS activity in the C3, C3–C4 intermediate, and C4 leaves, respectively. It had similar elution characteristics to the cytosolic GS from barley and was readily bound by the antibodies made against barley cytosolic GS (GS1). This enzyme, identified as GS1, was thought to be cytosolic in origin. The results demonstrate that even within a single genus, the GS isoform composition is closely related to the type of photosynthetic pathway present within the leaf tissue.

Glutamine Synthetases of Higher Plants

The chromatographic properties of glutamine synthetase isoforms have been investigated in a wide range of higher plant leaves and shoots using ion exchange chromatography. Different patterns of glutamine synthetase isoform content were observed. Among higher plants, four patterns or groups could be recognized. The first group is characterized by having only cytosolic glutamine synthetase, whereas the second group is distinguished by having only chloroplastic glutamine synthetase. The third group is characterized by cytosolic glutamine synthetase being a minor component of the total leaf glutamine synthetase activity. The fourth group is distinct from the other groups in having high cytosolic and chloroplast glutamine synthetase activity. Immunological studies have been undertaken on a few species from each group to identify unambiguously both cytosolic and chloroplastic glutamine synthetases.

Developmental Formation of Glutamine Synthetase in Greening Pumpkin Cotyledons and Its Subcellular Localization

During the germination of pumpkin (Cucurbita sp. Amakuri Nankin) seeds in dark, the activity of glutamine synthetase in cotyledons gradually increased, reaching a maximum at 5 to 6 days. A measurable enhancement (about 4-fold) of the enzyme activity occurred when the seedlings were exposed to continuous illumination from day 4 up to day 8. Glutamine synthetase activity was detectable only in the cytosolic fraction in the etiolated cotyledons, whereas it was found both in the cytosolic and chloroplast fractions in the green cotyledons. The two isoenzymes of glutamine synthetase have been separated by DEAE-cellulose column chromatography of extracts from the green cotyledons. These data indicate that during the greening process the chloroplastic glutamine synthetase is newly synthesized. The roles of cytosolic and chloroplastic glutamine synthetase in germinating pumpkin cotyledons concerning assimilation of NH(3) are discussed.

Evidence for a cytosolic-dependent light induction of chloroplastic glutamine synthetase during greening of etiolated rice leaves

During the greening of etiolated rice leaves, total glutamine synthetase activity increases about twofold, and after 48 h the level of activity usually observed in green leaves is obtained. A density-labeling experiment with deuterium demonstrates that the increase in enzyme activity is due to a synthesis of the enzyme. The enhanced activity obtained upon greening is the result of two different phenomena: there is a fivefold increase of chloroplastic glutamine synthetase content accompanied by a concommitant decrease (twofold) of the cytosolic glutamine synthetase. The increase of chloroplastic glutamine synthetase (GS2) is only inhibited by cycloheximide and not by lincomycin. This result indicates a cytosolic synthesis of GS2. The synthesis of GS2 was confirmed by a quantification of the protein by an immunochemical method. It was demonstrated that GS2 protein content in green leaves is fivefold higher than in etiolated leaves.

Effect of light on the formation of multiple molecular forms of glutamine synthetase in plants.

The presence of multiple molecular forms (MMF) of glutamine synthetase (GS) has been studied in pumpkin plants and in cotyledons of bean plants. Two MMF of GS have been found in pumpkin leaves and in green cotyledons: chloroplast GS and cytosol GS. Cotyledons of etiolated pumpkin seedlings contain only the cytosol GS. Illumination of etiolated pumpkin seedlings with white light results in the appearance, within one minute, of the second molecular form, the chloroplast GS, which appears to be due to activation rather than de novo synthesis of the enzyme. Cotyledons of resting seeds of horse bean, pea, soybean and lupine contain only one form of GS. The second form, chloroplast GS, appears after germination in the light, but only in those cotyledons of soybean and lupine that can become green.

Identification of multiple forms of glutamine synthetase in field bean ( Vicia faba L.)

Glutamine synthetase (GS) activity was detected after electrophoresis on starch gels using the glutamyl transfer reaction. Two bands of GS activity (designated GS 1 and GS 2) were observed in a chloroplast fraction and four bands (designated GS 1, GS 2, GS 4 and GS 5) in a total leaf extract from field beans. GS 4 and GS 5 were assumed to be cytoplasmic GS. Differences in the chloroplast GS bands (GS 1 and GS 2) but not in the cytoplasmic bands (GS 4 and GS 5) were observed between cultivars and between lines of the same cultivar. No differences in GS banding patterns were noted between extracts from pods or subtended leaves sampled at different stages of development. Other field bean plants were grown from seed in perlite moistened with distilled water and then pretreated with solutions of NaNO 3, NH 4NO 3, NH 4Cl or urea for either 1 or 7 days. Electrophoresis of root extracts from plants pretreated for 1 day with NH 4NO 3, NH 4Cl or urea revealed the presence of 1 band of GS activity, designated GS 3, clearly distinguished from the 2 forms in the leaf cytoplasm. An additional band with a mobility similar to one of those in the chloroplast, GS 1, was found in root extracts of plants given NaNO 3 or NH 4NO 3 for 1 week, but not in those pretreated with NH 4Cl or urea. Incubation of the starch gel with methionine sulphoximine (MSO) in the presence of ATP and Mg 2+ prior to the detection of GS activity prevented any bands being observed. The possible roles of these different forms of GS are discussed in the light of previously reported evidence.

Electron microscopy of glutamine synthetase from pea leaf chloroplasts

The structure of pea leaf chloroplast glutamine synthetase was studied by electron microscopy. The enzyme is shown to consist of eight elongated subunits which are arranged with point 42 symmetry at the vertices of two squares. These squares are twisted about the 4-fold axis at 40 degrees relative to each other.