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.

     

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.

     

Subunit Composition of Glutamine Synthetase Isozymes from Root Nodules of Bean (Phaseolus vulgaris L.)

Glutamine synthetase from bean nodules can be separated into two isoforms, GS_n1 and GS_n2. A purification protocol has been developed. It included protamine sulfate precipitation, ammonium sulfate fractionation, anthranilate-affinity chromatography, Dye-Matrex (Orange A) chromatography, and diethylaminoethyl-cellulose ion-exchange chromatography. GS_n1 and GS_n2 have been purified to homogeneity. Subunit structure analysis using two-dimensional polyacrylamide gel electrophoresis revealed that GS_n1 was composed of two different types of subunit polypeptides. They differed in isoelectric points (6.0 and 6.3) but had the same molecular weights (46,000 Daltons). GS_n2 was composed of only one type of subunit polypeptide. It had an isoelectric point of 6.0 and a molecular weight of 46,000 Daltons. It was apparently identical to one of the polypeptides found in GS_n1. Glutamine synthetase holoenzyme consisted of eight subunits. In the nodule there are two different types of glutamine synthetase subunit polypeptides. Random combinations of the polypeptides should generate nine different isozymes. Our electrophoretic analysis revealed that GS_n2 was but one of the isozymes, and GS_n1 was a composite of the other eight. Hence, nodule glutamine synthetase isozymes were homo-octameric as well as hetero-octameric.     

Nitrogen Assimilating Enzyme Activities and Enzyme Protein during Development and Senescence of Effective and Plant Gene-Controlled Ineffective Alfalfa Nodules

Effective (N₂-fixing) alfalfa (Medicago sativa L.) and plant-controlled ineffective (non-N₂-fixing) alfalfa recessive for the in₁ gene were compared to determine the effects of the in₁ gene on nodule development, acetylene reduction activity (ARA), and nodule enzymes associated with N assimilation and disease resistance. Effective nodule ARA reached a maximum before activities of glutamine synthetase (GS), glutamate synthase (GOGAT), aspartate aminotransferase (AAT), asparagine synthetase (AS), and phosphoenolpyruvate carboxylase (PEPC) peaked. Ineffective nodule ARA was only 5% of effective nodule ARA. Developmental profiles of GS, GOGAT, AAT, and PEPC activities were similar for effective and ineffective nodules, but activities in ineffective nodules were lower and declined earlier. Little AS activity was detected in developing ineffective nodules. Changes in GS, GOGAT, AAT, and PEPC activities in developing and senescent effective and ineffective nodules generally paralleled amounts of immunologically detectable enzyme polypeptides. Effective nodule GS, GOGAT, AAT, AS, and PEPC activities declined after defoliation. Activities of glutamate dehydrogenase, malate dehydrogenase, phenylalanine ammonia lyase, and caffeic acid-o-methyltransferase were unrelated to nodule effectiveness. Maximum expression of nodule N-assimilating enzymes appeared to require the continued presence of a product associated with effective bacteroids that was lacking in in₁ effective nodules.     

Isolation and Immunochemical Characterization of Plant Glutamine Synthetase in Alfalfa (Medicago sativa L.) Nodules

Host plant glutamine synthetase (GS) has been purified 100-fold from N₂-fixing alfalfa (Medicago sativa L.) nodules by a new procedure involving preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as a final step. An SDS-polypeptide fraction corresponding to plant GS was identified and consisted of two major polypeptides of 40,000 to 45,000 molecular weight. Antibodies to the SDS-polypeptide fraction were raised in mice by intraperitoneal injection, and antisera were collected as ascitic fluid. Crude extracts of soluble protein from the plant fraction of nodules were resolved by SDS-PAGE and then subjected to electrophoresis in the second dimension into antibody-containing agarose gel. A single immunochemically active protein species was observed using this crossed immunoelectrophoresis method, even though both major GS SDS-polypeptides were apparently resolved in the first (SDS-PAGE) dimension. Plant GS protein in crude nodule extracts was quantitated immunochemically by comparison with immunoprecipitin arcs of similarly treated amounts of pure antigen. Using this technique, it was determined that plant GS was present at 150 micrograms per gram fresh weight or 1.2% of total plant soluble protein in N₂-fixing alfalfa nodules.

Results suggest that alfalfa nodule plant GS consists of two major subunit polypeptides, but only a single immunochemically active native protein was observed. The crossed immunoelectrophoresis procedure described here should be generally applicable for immunochemical detection of lower abundance components of crude plant extracts.

     

Purification and properties of glutamine synthetase from the plant cytosol fraction of lupin nodules

Glutamine synthetase from the plant cytosol fraction of lupin nodules was purified 89-fold to apparent homogeneity. The enzyme molecule is composed of eight subunits of M_r 44,700 ± 10%. Kinetic analysis indicates that the reaction mechanism is sequential and there is some evidence that Mg-ATP is the first substrate to bind to the enzyme. Michaelis constants for each substrate using the ammonium-dependent biosynthetic reaction are as follows: ATP, 0.24 mm; l-glutamate, 4.0–4.2 mm; ammonium, 0.16 mm. Using an hydroxamate-forming biosynthetic reaction the K_m ATP is 1.1 mm but the K_m for l-glutamate is not altered. The effect of pH on the K_m for ammonium indicates that NH₃ rather than NH₄⁺ may be the true substrate. At 10 mm Mg²⁺, the pH optimum of the enzyme is between 7.5 and 8, but increasing Mg²⁺ concentrations produce progressively more acidic optima while lower Mg²⁺ concentrations raise the pH optimum. The rate-response curve for Mg²⁺ is sigmoidal becoming bell-shaped in alkaline conditions. The enzyme is inhibited by l-Asp (K_i, 1.4 mm) and less markedly by l-Gln and l-Asn. Inhibition by ADP and AMP is strong, both nucleotides exhibiting K_i values around 0.3 mM. Investigations of the probable physiological conditions within the nodule plant cytosol indicate that in situ glutamine synthetase has an activity greater than that required to support the efflux of amino acid nitrogen from the nodule. A possible role for glutamine synthetase in the control of nodule ammonium assimilation is suggested.     

Appearance of a novel form of plant glutamine synthetase during nodule development in Phaseolus vulgaris L.

The activities of glutamine synthetase (GS), nitrogenase and leghaemoglobin were measured during nodule development in Phaseolus vulgaris infected with wild-type or two non-fixing (Fix^-) mutants of Rhizobium phaseoli. The large increase in GS activity which was observed during nodulation with the wild-type rhizobial strain occurred concomitantly with the detection and increase in activity of nitrogenase and the amount of leghaemoglobin. Moreover, this increase in GS was found to be due entirely to the appearance of a novel form of the enzyme (GS_n1) in the nodule. The activity of the form (GS_n2) similar to the root enzyme (GS_r) remained constant throughout the experiment. In nodules produced by infection with the two mutant strains of Rhizobium phaseoli (JL15 and JL19) only trace amounts of GS_n1 and leghaemoglobin were detected.Abbreviations DEAE-Sephacel diethylaminoethyl-Sephacel - GS glutamine synthetase     

Immunochemical comparison of glutamine synthetases from some solanaceae plants

The presence of different glutamine synthetase isoenzymes in different Solanaceae plants and their relative antigenicities against antiglutamine synthetase from tomato leaf serum were studied. All the plants tested showed one glutamine synthetase isoenzyme except for Mandragora autumnalis, which showed two, after discontinuous polyacrylamide gel electrophoresis and specific in situ assay. Antigenicities were compared by the double immunodiffusion technique. The Nicotiana glauca enzyme showed equal reactivity to that of Lycopersicon esculentum, but its antigenicity was higher than Withania frutescens, Datura stramonium, and Hyoscyamus niger. The study of relative antigenicities permitted differentiation of the glutamine synthetase enzymes from uncultivated species of Solanaceae.     

Antipeptide Antibodies That Can Distinguish Specific Subunit Polypeptides of Glutamine Synthetase from Bean (Phaseolus vulgaris L.)

The amino acid sequences of the β and γ subunit polypeptides of glutamine synthetase from bean (Phaseolus vulgaris L.) root nodules are very similar. However, there are small regions within the sequences that are significantly different between the two polypeptides. The sequences between amino acids 2 and 9 and between 264 and 274 are examples. Three peptides (γ_2-9, γ_264-274, and β_264-274) corresponding to these sequences were synthesized. Antibodies against these peptides were raised in rabbits and purified with corresponding peptide-Sepharose affinity chromatography. Western blot analysis of polyacrylamide gel electrophoresis of bean nodule proteins demonstrated that the anti-β_264-274 antibodies reacted specifically with the β polypeptide and the anti-γ_264-274 and anti-γ_2-9 antibodies reacted specifically with the γ polypeptide of the native and denatured glutamine synthetase. These results showed the feasibility of using synthetic peptides in developing antibodies that are capable of distinguishing proteins with similar primary structures.