Glutamate synthase isoforms in rice: immunological studies of enzymes in green leaf, etiolated leaf, and root tissues

Rabbit antiserum was raised against ferredoxin-dependent glutamate synthase (EC 1.4.7.1) purified from green leaves of Oryza sativa L. cv Delta. Ferredoxin-dependent glutamate synthase, detected in green leaf, etiolated leaf, and root tissues cross-reacted completely with the antiferredoxin glutamate synthase immunoglobulin G. In contrast, the immunoglobulin G did not cross-react with NADH-dependent (EC 1.4.1.14) and NADPH-dependent (EC 1.4.1.13) glutamate synthases found in nonphotosynthetic etiolated leaf and root tissues. In addition, ferredoxin-dependent glutamate synthase was separated and distinguished by its affinity to ferredoxin from NAD(P)H-dependent glutamate synthase on ferredoxin-Sepharose affinity chromatography. Based on the immunological studies, it is suggested that ferredoxin-dependent glutamate synthases in green leaf and etiolated leaf tissues are closely related proteins; in contrast, ferredoxin-dependent glutamate synthase in root tissue is a distinct protein from the leaf enzymes.     

Glutamate synthase in Medicago sativa L. Occurrence and properties of FD-dependent enzyme in plant cell fraction during root nodule development

In the plant cell fraction of Medicago sativa (L. cv Europe) nodules, glutamate synthase is active with reduced Fd, MV, NADH and NADPH as an electron donor. Up to 25 to 30 days after inoculation, the activities of Fd-dependent glutamate synthase (EC 1.4.1.7), the most active form of the enzyme, NADH-dependent (EC 1.4.1.14) and NADPH-dependent (EC 1.4.1.13) glutamate synthases increase about 2-fold followed by a relatively constant level per gram fresh weight of nodules. The activities of glutamate synthases with different electron carriers increase constantly about 30-fold after 46 days of inoculation by total fresh weight of nodules per plant. These nodule glutamate synthase activities with Fd, NADH or NADPH represent 30% relative to those of root glutamate synthases per plant with the respective electron donor. Fd-glutamate synthase in nodule plant fraction is a protein molecule immunochemically distinct from pyridine nucleotide-glutamate synthases. MV-linked enzyme activity is associated with Fd-glutamate synthase. The Fd-glutamate synthase has a subunit molecular mass of 68.2 kDa, and it exhibits a high affinity for spinach Fd as an electron carrier. The increase in Fd-glutamate synthase activity during nodule development is accompanied by a rise in the enzyme protein content. The total activity of different forms of glutamate synthase in vitro ensures a higher level than the rate of ammonia production during N2 fixation in bacteroids of Medicago sativa nodules.     

Occurrence of ferredoxin-dependent glutamate synthase in plant cell fraction of soybean root nodules (Glycine max)

Ferredoxin-dependent glutamate synthase (EC 1.4.7.1) and NADH-dependent glutamate synthase (EC 1.4.1.14) have been identified in the plant cells of soybean nodules. Ferredoxin-dependent glutamate synthase is 2-fold more active than NADH-dependent enzyme in vitro. Ferredoxin-dependent glutamate synthase cross-reacts with IgG against ferredoxin-dependent glutamate synthase of rice green leaves, whereas NADH-dependent glutamate synthase does not recognize the IgG, indicating that there are two distinct enzyme proteins. Ferredoxin-dependent glutamate synthase is composed of polypeptide chain(s) of 165 kDa and has a high affinity to spinach leaf ferredoxin as an electron carrier.     

Location of two isoenzymes of NADH-dependent glutamate synthase in root nodules of Phaseolus vulgaris L.

The two isoenzymes of NADH-dependent glutamate synthase (NADH-GOGAT; EC 1.4.1.14), previously identified in root nodules of Phaseolus vulgaris L., have both been shown to be located in root-nodule plastids. The nodule specific NADH-GOGAT II accounts for the majority of the activity in root nodules, and is present almost exclusively in the central tissue of the nodule. However about 20% of NADH-GOGAT I activity is present in the nodule cortex, at about the same specific activity as this isoenzyme is found in the central tissue. Glutamine synthetase (GS; EC 6.3.1.2) occurs predominantly as the polypeptide in the central tissue, whereas in the cortex, the enzyme is represented mainly by the polypeptide. Over 90% of both GS and NADH-GOGAT activities are located in the central tissue of the nodule and GS activity exceeds NADH-GOGAT activity by about twofold in this region. Using the above information, a model for the subcellular location and stoichiometry of nitrogen metabolism in the central tissue of P. vulgaris root nodules is presented.Abbreviations Fd-GOGAT ferredoxin-dependent glutamate synthase - GOGAT glutamate synthase - GS glutamine synthetase - NADH-GOGAT NADH-dependent glutamate synthase - IEX-HPLC ion-exchange high-performance liquid chromatography     

Tissue distribution and developmental patterns of NADH-dependent and ferredoxin-dependent glutamate synthase activities in maize (Zea mays) kernels

Both NADH-dependent glutamate synthase (NADH-GOGAT, EC 1.4.1.14) and ferredoxin-dependent glutamate synthase (Fd-GOGAT, EC 1.4.7.1) activities were present in the endosperm, embryo, pedicel and pericarp of maize ( Zea mays L. var. W64A × A619) kernels. The endosperm contained the highest proportions of each activity on a per tissue basis. In the endosperm, NADH-GOGAT and Fd-GOGAT activities increased 12- and 2.5-fold, respectively, during early zein accumulation. NADH-GOGAT and Fd-GOGAT activities were expressed in the upper, middle and lower portions of the endosperm in a manner that paralleled but preceded zein accumulation. Maize endosperm NADH-GOGAT was purified 159-fold using ammonium sulfate fractionation, anion exchange chromatography and dye-ligand chromatography. Apparent K_m values for glutamine, α-ketoglutarate and NADH were 850, 19 and 1 μM, respectively. The results are consistent with endosperm GOGAT functioning to redistribute nitrogen from glutamine, the predominant nitrogenous compound delivered to the endosperm, into other amino acids needed for storage protein synthesis.     

NaCl effects on proline metabolism in rice (Oryza sativa) seedlings

Salt-stress effects on osmotic adjustment, ion and proline concentrations as well as proline metabolizing enzyme activities were studied in two rice ( Oryza sativa L.) cultivars differing in salinity resistance: I Kong Pao (IKP; salt-sensitive) and Nona Bokra (salt-resistant). The salt-sensitive cultivar exposed to 50 and 100 m M NaCl in nutritive solution for 3 and 10 days accumulated higher levels of sodium and proline than the salt-resistant cultivar and displayed lower levels of osmotic adjustment. Proline accumulation was not related to proteolysis and could not be explained by stress-induced modifications in Δ¹-pyrroline-5-carboxylate reductase (P5CR; EC 1.5.1.2) or proline dehydrogenase (PDH; EC 1.5.1.2) activities recorded in vitro. The extracted ornithine Δ -aminotransferase (OAT; EC 2.6.1.13) activity was increased by salt stress in the salt-sensitive cultivar only. In both genotypes, salt stress induced an increase in the aminating activity of root glutamate dehydrogenase (GDH; EC 1.4.1.2) while deaminating activity was reduced in the leaves of the salt-sensitive cultivar. The total extracted glutamine synthetase activity (GS; EC 6.3.1.2) was reduced in response to salinity but NaCl had contrasting effects on GS1 and GS2 isoforms in salt-sensitive IKP. Salinity increased the activity of ferredoxin-dependent glutamate synthase (Fd-GOGAT; EC 1.4.7.1) extracted from leaves of both genotypes and increased the activity of NADH-dependent glutamate synthase (NADH-GOGAT; EC 1.4.1.14) in the salt-sensitive cultivar. It is suggested that proline accumulation is a symptom of salt-stress injury in rice and that its accumulation in salt-sensitive plants results from an increase in OAT activity and an increase in the endogenous pool of its precursor glutamate. The physiological significance of the recorded changes are analyzed in relation to the functions of these enzymes in plant metabolism.     

Purification and characterization of mitochondrial citrate synthase

Mitochondrial citrate synthase was purified from leaves of Pisum sativum L. cv Progress 9. A three step purification was employed using ATP-Sepharose affinity chromatography which resulted in a 600-fold enrichment. Enzyme activity was assayed spectrophotometrically during greening of etiolated leaves under constant white light illumination. An increase (1.4 fold) in citrate synthase activity was observed in response to light. Immunoblot analysis of the same samples indicated a constant steady state level of citrate synthase on a per milligram protein basis. These investigations provide supportive evidence for the ability of this trichloroacetic acid cycle enzyme to be active in photosynthesizing tissue.     

Antigenic similarities between ferredoxin-dependent nitrite reductase and glutamate synthase fromChlamydomonas reinhardtii

Polyclonal antisera were prepared against ferredoxin-nitrite reductase (EC 1.7.7.1) and ferredoxin-glutamate synthase (glutamate synthase (ferredoxin); EC 1.4.7.1) from the green algaChlamydomonas reinhardtii. The anti-glutamate synthase antibodies recognized both glutamate synthase and nitrite reductase, but inhibited only the ferredoxin-linked activity of the latter enzyme and not the activity dependent on methyl viologen. Analogously, the anti-nitrite reductase antibodies recognized glutamate synthase and nitrite reductase but the first enzyme was only poorly inhibited. Free ferredoxin protected the nitrite reductase against its inactivation by anti-glutamate synthase antibodies. These results indicate that the ferredoxin-dependent glutamate synthase and nitrite reductase from this alga share common antigenic determinants, and that these are located at the ferrodoxin-binding domains.     

The development of NAD(P)H-dependent and ferredoxin-dependent glutamate synthase in greening barley and pea leaves

The activity of NAD(P)H-dependent glutamate synthase (E.C. 1.4.1.14) has been demonstrated in extracts from etiolated shoots of pea (Pisum sativum L.) and barley (Hordeum vulgare L.). This activity does not significantly alter upon greening of the etiolated shoots, and is at a similar level in light-grown material. Ferredoxin-dependent glutamate synthase (E.C. 1.4.7.1) has low activity in etiolated shoots but increases rapidly on greening. In light grown leaves ferredoxin-dependent activity is 30–40-fold higher than NAD(P)H-dependent activity. It is not considered that the NAD(P)H-dependent glutamate synthase plays an important role in ammonia assimilation in the photosynthetic tissue of higher plants.     

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.     

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

Seasonal 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.     

Localization of Nitrogen-Assimilating Enzymes in the Chloroplast of Chlamydomonas reinhardtii

The specific activities of nitrate reductase, nitrite reductase, glutamine synthetase, glutamate synthase, and glutamate dehydrogenase were determined in intact protoplasts and intact chloroplasts from Chlamydomonas reinhardtii. After correction for contamination, the data were used to calculate the portion of each enzyme in the algal chloroplast. The chloroplast of C. reinhardtii contained all enzyme activities for nitrogen assimilation, except nitrate reductase, which could not be detected in this organelle. Glutamate synthase (NADH- and ferredoxin-dependent) and glutamate dehydrogenase were located exclusively in the chloroplast, while for nitrite reductase and glutamine synthetase an extraplastidic activity of about 20 and 60%, respectively, was measured. Cells grown on ammonium, instead of nitrate as nitrogen source, had a higher total cellular activity of the NADH-dependent glutamate synthase (+95%) and glutamate dehydrogenase (+33%) but less activity of glutamine synthetase (−10%). No activity of nitrate reductase could be detected in ammonium-grown cells. The distribution of nitrogen-assimilating enzymes among the chloroplast and the rest of the cell did not differ significantly between nitrate-grown and ammonium-grown cells. Only the plastidic portion of the glutamine synthetase increased to about 80% in cells grown on ammonium (compared to about 40% in cells grown on nitrate).     

Phytochrome mediated regulation of sucrose phosphate synthase activity in maize

The extractable activity of sucrose phosphate synthase was determined in etiolated seedlings of maize (Zea mays L.), soybean (Glycine max [L.] Merr.), and sugar beet (Beta vulgaris L.) following treatments of changing light quality. A 30-minute illumination of 30 microeinsteins per square meter per second white light produced a three-fold increase in sucrose phosphate synthase activity at 2 hours postillumination when compared to seedlings maintained in total darkness. Etiolated maize seedlings treated with 3.6 microeinsteins per square meter per second of red and far-red light showed a 50% increase and a 50% decrease in sucrose phosphate synthase activity, respectively, when compared to etiolated maize seedlings treated with white light. Maize seedlings exposed for 30 minutes to red followed by 30 minutes to far-red showed an initial increase in sucrose phosphate synthase activity followed by a rapid decrease to control level. Neither soybean or sugar beet sucrose phosphate synthase responded to the 30-minute illumination of white light. Phytochrome is involved in sucrose phosphate synthase regulation in maize, whereas it is not responsible for changes in sucrose phosphate synthase activity in soybean or sugar beet.     

Purification, Characterization, and Immunological Properties of NADH-Dependent Glutamate Synthase from Rice Cell Cultures

To obtain a monospecific antibody against NADH-dependent glutamate synthase (NADH-GOGAT; EC 1.4.1.14), the enzyme was purified to homogeneity from cultured rice cells (Oryza sativa) with column chromatography using Butyl Toyopearl 650M, Sephacryl S-300, Blue Sepharose CL-6B, and Butyl Toyopearl 650S. The specific activity at the final stage of the purification was 9.8 micromoles of glutamate formed per minute per milligram of protein. The yield was 6.1% and purification was 815-fold. Analysis by denaturing gel electrophoresis revealed a single polypeptide with an apparent molecular weight of 196,000, similar to the value of 194,000 estimated for the native protein. Apparent K_m values for l-glutamine, 2-oxoglutarate, and NADH were 811, 76, and 3.0 micromolar, respectively. Neither NADPH nor l-asparagine substituted for NADH and l-glutamine, respectively. The enzyme had its absorption maxima at 273, 373, and 440 nanometers with a shoulder at 475 nanometers, suggesting that the rice NADH-GOGAT is a flavoprotein. Monospecific antibody raised against NADH-GOGAT purified from the rice cells was obtained as the first instance for the enzyme in higher plants. Immunological analyses showed that the antibody for rice cell NADH-GOGAT reacted with only the enzyme in extracts from the cells. The anti-NADH-GOGAT antibody did not recognize the ferredoxin-GOGAT purified from rice leaves, and likewise the anti-rice leaf ferredoxin-GOGAT antibody did not react with the NADH-GOGAT purified from the cultured rice cells.     

Regulation of Glutamine Synthetase by Light and during Nitrogen Deficiency in Synchronous Chlorella sorokiniana

A method is described to achieve density labeling of proteins in unicellular algae by using ¹³CO₂. This is a satisfactory procedure especially for work on nitrogen metabolism. The increase in activity of glutamine synthetase (EC 6.3.1.2.) and glutamate synthase (EC 1.4.7.1.) in Chlorella sorokiniana mediated by a dark/light shift and by nitrogen starvation were investigated. Using the method of density labeling and isopycnic centrifugation, we demonstrated that the increase in enzyme activity after a dark/light shift is based on activation rather than de novo synthesis. The increase in enzyme activity after transfer to nitrogen-deficient medium is based both on activation and de novo synthesis.     

Biochemical, electrophoretic and immunological characterization of peroxisomal enzymes in three soybean organs

Biochemical, electrophoretic and immunological studies were made among peroxisomal enzymes in three organs of soybean [Glycine max (L.) Merr. cv. Centennial] to compare the enzyme distribution and characteristics of specialized peroxisomes in one species. Leaves, nodules and etiolated cotyledons were compared with regard to several enzymes localized solely in their peroxisomes: catalase (EC 1.11.1.6), malate synthase (EC 4.1.3.2), glycolate oxidase (EC 1.1.3.1), and urate oxidase (EC 1.7.3.3). Catalase activity was found in all tissue extracts. Electrophoresis on native polyacrylamide gels indicated that leaf catalase migrated more anodally than nodule or cotyledon catalase as shown by both activity staining and Western blotting. Malate synthase activity and immunologically detectable protein were present only in the cotyledon extracts. Western blots of denaturing (lithium dodecyl sulfate) gels probed with anti-cotton malate synthase antiserum, reveal a single subunit of 63 kDa in both cotton and soybean cotyledons. Glycolic acid oxidase activity was present in all three organs, but ca 20-fold lower (per mg protein) in both nodule and cotyledon extracts compared to leaf extracts. Electrophoresis followed by activity staining on native gels indicated one enzyme form with the same mobility in nodule, cotyledon and leaf preparations. Urate oxidase activity was found in nodule extracts only. Native gel electrophoresis showed a single band of activity. Novel electrophoretic systems had to be developed to resolve the urate oxidase and glycolate oxidase activities; both of these enzymes moved cathodally in the gel system employed while most other proteins moved anodally. This multifaceted study of enzymes located within three specialized types of peroxisomes in a single species has not been undertaken previously, and the results indicate that previous comparisons between the enzyme content of specialized peroxisomes from different organisms are mostly consistent with that for a single species, soybean.     

Intracellular distribution of enzymes associated with nitrogen assimilation in roots

The cellular distribution of enzymes involved in nitrogen assimilation: nitrate reductase (EC 1.6.6.2), nitrite reductase (EC 1.6.6.4), glutamine synthetase (EC 6.3.1.2), glutamate synthase (EC 2.6.1.53), and glutamate dehydrogenase (EC 1.4.1.3) has been studied in the roots of five plants: maize (Zea mays L. hybrid W 64A x W 182E), rice (Oryza sativa L. cv. Delta), bean (Phaseolus vulgaris L. cv. Contender), pea (Pisum sativum L. cv. Demi-nain), and barley (Hordeum vulgare L.). Initially, cell organelles were separated from soluble proteins by differential centrifugation. Cell organelles were also subjected to sucrose density gradients. The results obtained by these two methods indicate that nitrite reductase and glutamate synthase are localized in plastids, nitrate reductase and glutamine synthetase are present in the cytosol, and glutamate dehydrogenase is a mitochondrial enzyme.     

Salt stress increases ferredoxin-dependent glutamate synthase activity and protein level in the leaves of tomato

Ferredoxin-dependent glutamate synthase (EC 1.4.7.1) catalyzes an essential step in the pathway of glutamate biosynthesis. Exposing detached tomato ( Lycopersicon esculentum ) leaves for 6 h to 12 g l⁻¹ NaCl resulted in a significant two-fold increase in the activity of ferredoxin-dependent glutamate synthase extracted from the leaves. Western blot studies demonstrated that salt treatment also increased the ferredoxin-dependent glutamate synthase content of the leaves. A similar effect of salt on the concentration of this enzyme was found in the leaves of hydroponically-grown tomato plants. The induction of ferredoxin-dependent glutamate synthase under salt stress may provide the glutamate required for the proline synthesis which is a common response to salt stress.     

Regulation of the ammonia assimilatory enzymes in Salmonella typhimurium.

The regulation of glutamate dehydrogenase (EC 1.4.1.4), glutamine synthetase (EC 6.3.1.2), and glutamate synthase (EC 2.6.1.53) was examined for cultures of Salmonella typhimurium grown with various nitrogen and amino acid sources. In contrast to the regulatory pattern observed in Klebsiella aerogenes, the glutamate dehydrogenase levels of S. typhimurium do not decrease when glutamine synthetase is derepressed during growth with limiting ammonia. Thus, it appears that the S. typhimurium glutamine synthetase does not regulate the synthesis of glutamate dehydrogenase as reported for K. aerogenes. The glutamate dehydrogenase activity does increase, however, during growth of a glutamate auxotroph with glutamate as a limiting amino acid source. The regulation of glutamate synthase levels is complex with the enzyme activity decreasing during growth with glutamate as a nitrogen source, and during growth of auxotrophs with either glutamine or glutamate as limiting amino acids.