Purification and physico-chemical properties of glutamine synthetase from pea chloroplasts]

A highly purified, practically homogeneous glutamine synthetase was isolated from pea leaf chloroplasts. The enzyme purity was assayed by polyacrylamide gel electrophoresis and analytical ultracentrifugation. The sedimentation coefficient is 16,3S. The sedimentation equilibrium analysis showed that the molecular weight of the enzyme is equal to 480 000. The minimal molecular weights of the enzyme as calculated from the data of polyacrylamide gel electrophoresis in the presence of SDS and the amino acid analysis were found to be 62 000 and 60 000, respectively. The enzyme contains a large amount of dicarboxylic and sulfur-containing amino-acids. The N-terminal amino acid is glycine. The isoelectric point for the enzyme lies within the pH range of 4,2-4-4.     

Glutamine Synthetase in Spinach Leaves : IMMUNOLOGICAL STUDIES AND IMMUNOCYTOCHEMICAL LOCALIZATION

By polyacrylamide gel electrophoresis, DEAE Sephacel, and hydroxyapatite chromatography, one form of glutamine synthetase has been identified in spinach (Spinacia oleracea L. cv. Monstrueux de Viroflay) leaves. It is localized only inside the chloroplast. The enzyme was purified to homogeneity and specific antibodies against the protein were raised by immunization of rabbits. The intracellular localization of glutamine synthetase in spinach leaves was studied by indirect immunofluorescence microscopy on thin-sectioned spinach leaves. It has been demonstrated that the enzyme is specifically associated with the chloroplasts of parenchymatous cells.     

Investigation of the site of synthesis of chIcoroplastic enzymes of nitrogen metabolism by the use of heat-treated 70S ribosomedeficient rye leaves

The activities of the enzymes nitrate reductase (EC 1.6.6.1), nitrite reductase (EC 1.6.6.4), glutamine synthetase (EC 6.3.1.2), glutamate synthase (GOGAT; EC 1.4.7.1), glutamate-oxaloacetate aminotransferase (EC 2.6.1.1), and glutamate dehydrogenase (EC 1.4.1.2) were compared in light-grown green or etiolated leaves of rye seedlings ( Secale cereale L. cv. Halo) raised at 22°C, and in the bleached 70S ribosome-deficient leaves of rye seedlings grown at a non-permissive high temperature of 32°C. Under normal permissive growth conditions the activities of most of the enzymes were higher in light-grown, than in dark-grown, leaves. All enzyme activities assayed were also observed in the heat-treated 70S ribosome-deficient leaves. Glutamine synthetase, glutamate synthase, and glutamate-oxaloacetate aminotransferase occurred in purified ribosome-deficient plastids separated on sucrose gradients. For glutamate-oxaloacetate aminotransferase four multiple forms were separated by polyacrylamide gel electrophoresis from leaf extracts. The chloroplastic form of this enzyme was also present in 70S ribosome-deficient leaves. It is concluded that the chloroplast-localized enzymes nitrite reductase, glutamine synthetase, glutamate synthase and glutamate-oxaloacetate aminotransferase, or their chloroplast-specific isoenzyme forms, are synthesized on cytoplasmic 80S ribosomes.     

Phenylalanyl-tRNA synthetase from chloroplasts of a higher plant (Phaseolus vulgaris). Purification and comparison of its structural, functional, and immunological properties with those of the enzymes from the corresponding cytoplasm, the cyanobacterium Anacystis nidulans, and the photosynthetic green sulfur bacterium Chlorobium limicola

Chloroplastic phenylalanyl-tRNA synthetase from bean leaves is purified under optimal protective conditions over 4,900-fold. Its apparent molecular weight is 78,000, as determined by gel filtration, with a dimeric subunit structure of alpha beta (alpha = 33,000 and beta = 42,000), as determined by sodium dodecyl sulfate gel electrophoresis. This indicates a drastic size reduction of 40% for each subunit compared to the corresponding cytoplasmic enzyme and a unique quaternary structure. Heterologous aminoacylation and substrate properties of ATP analogs indicate substantial differences in the topographies of the substrate binding domains of these two heterotopic intracellular plant enzymes. No common antigenic determinants with the bean cytoplasmic enzyme were detected by polyclonal antibodies against the chloroplastic enzyme. The same negative result applies to the immunological comparison with the partially purified enzymes from the cyanobacterium Anacystis nidulans and the photosynthetic green sulfur bacterium Chlorobium limicola that both have a molecular weight of 260,000.     

Two forms of glutamine synthetase in leaves of Cucurbita pepo

It has been shown that the leaves of pumpkin (Cucurbita pepo) contain two molecular forms of glutamine synthetase (GS), one occurring in the cytosol (GS₁)and the other in the chloroplasts (GS₂). The activities of both forms were greater when ammonium ion was infiltrated into the leaves and this was shown to be due to de novo synthesis. The two synthetases were purified by ammonium sulphate fractionation, ion exchange chromatography on DEAE-cellulose, selective adsorption on calcium phosphate gel, and preparative polyacrylamide gel electrophoresis. The MWs of GS₁ and GS₂, estimated by gel filtration on Sephacryl S-200, were 480 000 and 370 000 respectively. During polyacrylamide gel electrophoresis in the presence of SDS both GS₁ and GS₂ were dissociated into polypeptide chains with MWs of 58 000 and 50 000 respectively, suggesting that GS, ₁ and GS₂ are octamers consisting of identical monomers. The synthetases showed noticeable differences in their amino acid composition. In GS₁ and GS₂ the proportions of α- helical segments were 34 and 17 % respectively. In the presence of Mg²⁺, the pH optima for GS₁ and GS₂ were 7.25 and 7.75 respectively, and K_m values toward l-glutamate were 13 and 46 mM respectively. From the experimental data it is inferred that GS₁ and GS₂ are isoenzymes.     

Glutamine Synthetase in Rice: A COMPARATIVE STUDY OF THE ENZYMES FROM ROOTS AND LEAVES

Chromatographic, kinetic, and regulatory properties of glutamine synthetase in rice were investigated. By DEAE-Sephacel column chromatography, two forms (glutamine synthetase 1 and glutamine synthetase 2) were identified in leaves and one form (glutamine synthetase R) was identified in roots. Purification on hydroxyapatite and gel electrophoresis showed that glutamine synthetase R was distinct from the leaf enzymes. The three isoforms were purified to similar specific activities and their properties were studied. Heat lability, pH optimum about 8, K(m) for l-glutamate of 20 millimolar, and inhibition by glucosamine 6-phosphate were the main characteristics of glutamine synthetase 2. Heat stability, pH optimum about 7.5, K(m) for l-glutamate of 2 millimolar, and no effect of glucosamine 6-phosphate differentiated glutamine synthetase 1 from glutamine synthetase 2. Glutamine synthetase R was also a labile protein but its kinetic and regulatory properties were quite similar to those of glutamine synthetase 1. These results clearly demonstrate the existence of three isoforms of glutamine synthetase in rice, two of which are located in the leaves and the third in the roots.     

Glutamine synthetase of pseudomonads: some biochemical and physicochemical properties.

The glutamine synthetases from several Pseudomonas species were purified to homogeneity, and their properties were compared with those reported for the enzymes from Escherichia coli and other gram-negative bacteria. The glutamine synthetase from Pseudomonas fluorescens was unique because it was nearly precipitated quantitatively as a homogeneous protein during dialysis of partially purified preparations against buffer containing 10 mM imidazole (pH 7.0) and 10 mM MnCl2. The glutamine synthetases from Pseudomonas putida and Pseudomonas aeruginosa were purified by affinity chromatography on Affi-blue gel. Dodecamerous forms of the E. coli and P. fluorescens glutamine synthetases had identical mobilities during polyacrylamide gel electrophoresis. Their dissociated subunits, however, migrated differently and were readily separated by electrophoresis on polyacrylamide gels containing 0.1% sodium dodecyl sulfate. This difference in subunit mobilities is not related to the state of adenylylation. Regulation of the Pseudomonas glutamine synthetase activity is mediated by an adenylylation-deadenylylation cyclic cascade system. A sensitive procedure was developed for measuring the average number of adenylylated subunits per enzyme molecule for the glutamine synthetase from P. fluorescens. This method takes advantage of the large differences in transferase activity of the adenylylated and unadenylylated subunits at pH 6.0 and of the fact that the activities of both kinds of subunits are the same at pH 8.45.     

Methionyl-tRNA Synthetase from Phaseolus aureus: Purification and Properties

l-Methionyl-tRNA synthetase (EC 6.1.1.10) from seeds of Phaseolus aureus has been purified approximately 290-fold. Optimum assay conditions were determined by using the ATP-pyrophosphate exchange assay and the aminoacylation assay. The enzyme catalyzes both selenomethionine- and selenoethionine-dependent ATP-pyrophosphate exchange in addition to catalyzing the formation of selenomethionyl-tRNA at a rate comparable to the rate of formation of methionyl-tRNA. Competition experiments were conducted to investigate further the substrate specificity of the purified enzyme. Two peaks of methionyl-tRNA synthetase were detected by using Sephadex G-200 gel filtration; the molecular weights of the two enzymes as determined by Sephadex G-200 column chromatography were 340,000 and 85,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggests that the enzyme is a tetramer consisting of four identical monomers with molecular weights of 85,000.     

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

Summary 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.     

Purification and properties of hepatic glutamine synthetases from the ureotelic gulf toadfish, Opsanus beta

The cytoplasmic and mitochondrial forms of glutamine synthetase (GSase) were purified from the liver of the gulf toadfish Opsanus beta by modifications of methods previously applied to dogfish shark to examine their kinetic and structural properties. Both isozymes have subunit molecular weights of approximately 42 kDa (by SDS-PAGE) and native molecular weights of approximately 365 kDa (by gel filtration chromatography), suggesting an octomeric arrangement of the native enzymes. Identity of the purified proteins as GSase was further confirmed by western blot analysis using rabbit anti-chicken GSase antibodies. The requirement for MgCl₂ and several kinetic properties (e.g.,K_ms for glutamate, ATP and ammonia) of the two isozymes were very similar. Also notable was that both isozymes had K_ms for ammonia in the micromolar range (like the dogfish enzyme). These results suggest that the enzymes are probably easily saturated with ammonia under physiological conditions. The two GSase isozymes differed substantially in terms of inhibition by methionine sulfoximine, pH optima, specific activity and ratios of transferase to biosynthetic activities. Given the similarities in size, these results suggest that the molecular model of a single gene coding for both isozymes as has been demonstrated in the dogfish shark may not apply to the toadfish GSases.     

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.     

Purification and characterization of nucleoside diphosphate kinase from spinach leaves.

Two types of nucleoside diphosphate kinase (NDP kinase I and NDP kinase II) have been purified from spinach leaves to electrophoretic homogeneity. The enzymes were copurified with apparent [35S]GTP-gamma S-binding activities. NDP kinase I, which was not adsorbed to a hydroxyapatite column, and NDP kinase II, which was adsorbed, had molecular weights of 16,000 and 18,000, respectively, as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The molecular weights determined by gel filtration were 92,000 and 110,000, respectively, suggesting that both enzymes are composed of six identical subunits. Minor differences in some amino acids between NDP kinase I and NDP kinase II were observed when both enzymes were analyzed for amino acid composition. The apparent [35S]GTP gamma S-binding activity of purified NDP kinase I and NDP kinase II was found to be due to the formation of a [35S]thiophosphorylated enzyme, which is the intermediate of the NDP kinase reaction.     

Size and stability to sodium dodecyl sulfate of alkaline phosphatases from their three established human genes

Subunit molecular weights of human alkaline phosphatases (orthophosphoric-monoester phosphohydrolases (alkaline optimum), EC 3.1.3.1) determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS) were dependent upon acrylamide concentration, a reflection of their glycoprotein nature. Molecular weights at a concentration of 7% (w/w) or greater were 68300, 80800 and 79400 for the enzymes from placenta, liver and mucosa of small intestine, respectively. All enzymes were dimers, the respective native Mr values determined by gradient gel electrophoresis being 138000, 186000 and 180000. None of the molecular weights was altered by desialylation. Stability of the catalytic activity of the purified enzymes to SDS varied and was very dependent on pH. SDS at 1% (w/v) rapidly denatured both native and desialylated alkaline phosphatase from placenta at pH 7.5 but had little effect on these at pH 10.3. Compared with placenta, the native enzyme from liver had greater stability at pH 7.5 and both native and desialylated forms had lower stability at pH 10.3. The enzyme from intestinal mucosa was sharply different from the other two isoenzymes: SDS had little effect at pH 7.5 but very rapidly denatured the enzyme at pH 10.3. The size of alkaline phosphatases and their stability to SDS can be used to identify gene products and to recognize heterodimers formed between products of more than one gene.     

Characterization of alpha-Galactosidase from Cucumber Leaves

Two forms of alpha-galactosidase (alpha-d-galactoside galactohydrolase, E.C. 3.2.1.22) which differed in molecular weight were resolved from Cucumis sativus L. leaves. The enzymes were partially purified using ammonium sulfate fractionation, Sephadex gel filtration, and diethylaminoethyl-Sephadex chromatography. The molecular weights of the two forms, by gel filtration, were 50,000 and 25,000. The 50,000-dalton form comprised approximately 84% of the total alpha-galactosidase activity in crude extracts from mature leaves and was purified 132-fold. The partially purified 25,000-molecular weight form rapidly lost activity unless stabilized with 0.2% albumin and accounted for 16% of the total alpha-galactosidase activity in the crude extract. The smaller molecular weight form was not found in older leaves.The two forms were similar in several ways including their pH optima which were 5.2 and 5.5 for the 50,000- and 25,000-dalton form, respectively, and activation energies, which were 15.4 and 18.9 kilocalories per mole for the larger and smaller forms. Both enzymes were inhibited by galactose as well as by excess concentrations of p-nitrophenyl-alpha-d-galactoside sub-strate. K(m) values with this substrate and with raffinose and melibiose were different for each substrate, but similar for both forms of the enzyme. With stachyose, K(m) values were 10 and 30 millimolar for the 50,000- and 25,000- molecular weight forms, respectively.     

Peptidoglutaminase-I and II: Isolation in Homogeneous Form

Peptidoglutaminase-I and II that catalyzed the hydrolysis of the γ-amide of peptidebound glutamine, were purified from the cell-free extracts of Bacillus circulans by streptomycin sulfate precipitation, ammonium sulfate fractionation, DEAE-Sephadex, Sephadex G-200, QAE-Sephadex, hydroxylapatite-cellulose column chromatography, and finally preparative polyacrylantide gel disc electrophoresis. The purification steps resultd in a 714-fold increase in specific activity for peptidoglutaminase-I and in a 223-fold for peptidoglutaminase-II over the original extracts. The both enzymes were homogeneous in disc electrophoresis in polyacrylamide gel, immunoelectrophoresis in agar gel, and sedimentation analysis. Using gel filtration, the molecular weights of peptidoglutaminases I and II were estimated to be 90,000 and 125,000. However, during the purification steps, the both enzymes were observed to cause the dissociation and aggregation reaction which did not so much affect on their enzyme activities.     

Multiple molecular forms of glutamine synthetase in carp muscles]

Cytoplasmic and mitochondrial molecular forms of glutamine synthetase (CE 6.3.1.2) have been isolated from the carp muscle with purification degree of 100 and 165 times and output 9.0%. It is established that the temperature optimum of the cytoplasmic form activity is 30 degrees C and that of mitochondrial one--20 degrees C; the pH optimum for the both molecular forms is 6.0 and 8.2. The optimal ratio [Me2+] : [ATP] for the isolated form is 2:1; Km (seeming) of the cytoplasmic form in the presence of Mg2+ is 6.0 mM for glutamate, 0.035 for ammonium, for ATP 0.5 and 0.7 for magnesium ions; these values for the mitochondrial form are: 14.3, 0.048, 1.0 and 0.8 mM, respectively. Activity of the both glutamine synthetases with Mg2+ ions is almost by 50% higher than that of glutamine synthetases with Mn2+ ions. Seasonal regularities of the synthesis of molecular glutamine synthetase forms have been established in vivo. Cytoplasmic form is present in the muscles all year round, while mitochondrial one only in winter at low temperature of the environment and fish starvation. Differences in properties and seasonal character of synthesis of molecular glutamine synthetase forms in carp muscles are a result of diversity of their functional role.     

Multiple molecular forms of glutamine synthetase in pea seeds

Summary Multiple molecular forms of glutamine synthetase (GS, EC 6.3.1.2) have been studied in pea seeds of different varieties. The number of GS molecular forms in the seeds proved to be related to their colour. Two GS forms in the green seeds have been found and only one of them in the yellow seeds. Green seeds had chlorophyll content amounted to 0.4% of the total pigment content in the leaves. Chloroplasts, somewhat smaller than those in pea leaves of the same variety, have been isolated from green seeds. The presence of the second GS form in the pea green seeds we relate to the chloroplasts. By electrophoretic mobility both forms of GS from the green seeds are not identical to the chloroplast GS and the cytosol GS of leaves. Thus, we believe pea plant to contain, at least, four GS forms.     

Human deoxythymidine kinase. I. Purification and general properties of the cytoplasmic and mitochondrial isozymes derived from blast cells of acute myelocytic leukemia.

Two forms of deoxythymidine kinase from blast cells of acute myelocytic leukemia were identified by electrophoresis. One was associated mainly with the cytoplasm and the other with mitochondria. Both isozymes were separated and purified by differential affinity column chromatography which resulted in 2416- and 1634-fold purification of the cytoplasmic and mitochondrial enzymes, respectively. Affinity gel was prepared by linkage through position 3' of deoxythymidine. Each enzyme had the same electrophoretic mobility in the purified state as it did in the enzyme derived from the corresponding subcellular fraction of the homogenate. Thymidine phosphorylase was not retarded by the affinity column. The purified cytoplasmic and mitochondrial deoxythymidine kinase had different molecular weights, sensitivities to inhibition by ammonium sulfate, activation energies for the reaction and divalent cation requirements. Adenosine, guanosine, and cytosine 3':5'-monophosphates, putrescine, spermine, and spermidine were neither activators nor inhibitors of either deoxythymidine kinase.     

Chalcone synthases from spinach (Spinacia oleracea L.)

The two chalcone-synthase forms from leaves ofSpinacia oleracea L. were purified to apparent homogeneity. Antibodies were raised against both proteins in rabbits. The specificity of the antibodies was tested using immunotitration, immunoblotting, and immunoelectrophoresis techniques. The antibodies exhibited exclusive specificity for chalcone synthase and did not discriminate between the two antigens. The homodimeric chalcone synthases had the same subunit molecular weight but differed in their apparent native molecular weights. The peptide maps indicated extensive homology between the proteins. Chalcone-synthase activity was not detected in isolated spinach chloroplasts. Both enzyme forms were present in spinach cell-suspension cultures in which they were induced by light.Abbreviations DEAE diethylaminoethyl - DTE 1,4-dithioerythritol - EDTA ethylenediaminetetraacetic acid - HPLC high-performance liquid chromatography - IgG immunoglobulin G - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis Parts of the results were presented at the 14^th International Botanical Congress at Berlin in July 1987     

Secondary structure and functional groups of the active center of glutamine synthetase of pea chloroplasts

The circular dichroism spectra of glutamine synthetase (EC 6.3.1.2) from pea chloroplasts were recorded. Based on these spectra the percentage of alpha-helix sites, beta-structures, beta-bends and disordered sites of the polypeptide chain was calculated and was found equal to 23, 57, 1 and 23%, respectively. Data from protein photooxidation in the presence of methylene blue and the type of pH-dependence of pKm suggest that glutamate binding takes place on the imidazole ring of the histidine molecule. The inhibition of native glutamine synthetase by p-chloromercurybenzoate and the presence of free SH-groups in the enzyme molecule (approximately two SH-groups per monomer) suggest that these groups are the functional groups of the enzyme active center.