DOCSLIB.ORG

Measurement of in Vitro Glutamate Synthase Activity Using an Oxygen-Evolving Reconstituted Chloroplast System

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Measurement of in Vitro Glutamate Synthase Activity Using an Oxygen-Evolving Reconstituted Chloroplast System View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Volume 151, number 1 PEBS LETTERS January 1983 Measurement of in vitro glutamate synthase activity using an oxygen-evolving reconstituted chloroplast system Evidence for enzyme activation by Mg2+ and 2-oxoglutarate Ian B. Dry and Joseph T. Wiskich Department of Botany, University of Adelaide, Adelaide, SA 5000, Australia Received 5 November 1982 Glutamate synthase Reconstitution Chloroplast l14gz+ 2-Oxoglutarate Enzyme activation 1. INTRODUCTION 2. MATERIALS AND METHODS It is now evident that the major pathway for am- Pea seedlings (Pisum sativum var. Massey Gem) monia assimilation in plants involves the chloro- were grown in trays of vermiculite in a glasshouse plast enzyme, glutamate synthase (ECI4.7.1) [ 11. for lo-13 days. All biochemicals were purchased Despite its importance however, measurement of from Sigma. Spinach ferredoxin was prepared as in the in vitro activity of this enzyme, in the past, has PI. been time-consuming and somewhat complicated, Chloroplasts were isolated as in [9]. The chloro- involving the determination of the reaction pro- plast extract and envelope-free fractions were pre- duct glutamate [2-41. In order to allow for a more pared essentially as in [5] by resuspending the rapid and indeed more sensitive measurement of chloroplast pellet in 5.5 ml of a solution containing the in vitro activity of ferredoxin-dependent gluta- l/25 dilution of the standard reaction medium mate synthase, we have developed a reconstituted (330 mM sorbitol, 2 mM EDTA, 1 mM MgCl2, chloroplast system which exhibits GOGAT-depen- 1 mM MnC12, 50 mM Hepes-NaOH (pH 7.6)) dent 02 evolution. Such systems have been used to and 5 mM dithiothreitol, all adjusted to pH 7.6 study individual enzymes and enzyme sequences of with NaOH. After stirring for 2 min at 2°C the sus- the Benson-Calvin cycle [S-7]; however, this is the pension was centrifuged for 10 min at 14000 x g. first report of GOGAT-dependent 02 evolution in The supernatant (chloroplast extract) was collected a reconstituted chloroplast system. Furthermore, and the pellet resuspended in 40 ml of the same through the use of this assay system we have dis- medium and repelleted by centrifuging for 10 min covered regulatory characteristics not previously at 14000 x g. This pellet was then resuspended in reported for this enzyme. a small volume (2-3 ml) of full-strength reaction medium. Oxygen evolution was measured in a Rank 02 electrode maintained at 25°C and illuminated with Abbreviations: GOGAT, glutamate synthase; Mes, 2-(N- a 150 W tungsten halogen projector lamp giving a morpholino)-ethanesulfonic acid; Hepes, 4-(2-hydroxy- light intensity of 550 pE. mm2. SC’ at the centre of ethyl)-1-piperazineethane-sulfonic acid; BSA, bovine the vessel. A standard reaction mixture contained serum albumin; RuBP, ribulose 1,5-bisphosphate; envelope-free chloroplasts (200 pug chlorophyll), 2-OG, 2-oxoglutarate; Chl, chlorophyll chloroplast extract (50-200 ,ul), 330 mM sorbitol, Published by Elsevier Biomedical Press 00145793/83/OWO-0000ooo/$3.00 0 Federation of European Biochemical Societies 31 Volume 151, number 1 FEBS LETTERS January 1983 2 mM EDTA, 1 mM MnC12, 10 mM MgC12, mate produced in the GOGAT reaction, to initiate 50 mM Hepes-NaOH, 1 mM NHdCl, 600 units of any glutamine synthetase activity. The rate of 02 catalase, and 2OO/cg of spinach ferredoxin in a evolution achieved on a per mg chlorophyll basis in final volume of 2 ml at pH 7.6. the reconstituted system was dependent on the Chlorophyll was determined from 80% acetone amount of chloroplast extract added (fig. 2). In the extract as in [lo]. majority of experiments suboptimal levels of chloroplast extract were used to ensure that the supply of reducing equivalents from the chloro- 3. RESULTS phyll fraction, did not limit the rate of GOGAT The light-dependent evolution of 02 by a recon- catalysed 02 evolution, and consequently, the rates stituted chloroplast system in the presence of 2-OG given in the other figures do not represent the and glutamine is shown in fig. 1. It can be seen that maximum rates of 02 evolution which could have no 02 evolution occurs in the absence of either been achieved using this reconstituted system. substrate, and that evolution is inhibited by aza- The dependence of the reconstituted chloroplast serine, a specific inhibitor of the GOGAT enzyme system on added ferredoxin is illustrated in fig. 3. [2] and by DCMU which inhibits photosynthetic The K,,, for ferredoxin was 0.9 PM which compares electron transport. The rate of 02 evolution was in- favourably to the value of 2 PM as estimated in [2] sensitive to the addition of methionine sulfoximine for their enzyme. Some background activity was (not shown) indicating that the N&Cl present in the assay system, acted to uncouple electron trans- port only, and did not act together with the gluta- V I I I I I 50 100 I50 200 300 Chloroplast extract (~1) Fig. 1. GOGAT-dependent 02 evolution in a reconsti- Fig. 2. Effect of chloroplast extract on the rate of tuted chloroplast system. Both traces contained 125 ~1 of GOGAT-dependent 02 evolution in a reconstituted chloroplast extract as well as: (A) 4 mM glutamine; (B) chloroplast system. Assays contained 4 mM glutamine 1 mM 2-OG. Additions: (A) 2-OG, 1 mM; Azaserine, and 2 mM 2-oxoglutarate and the reaction mixtures were 0.5 mM; NADP, 0.16 mM; (B) glutamine, 4 mM; pre-incubated with both substrates and chloroplast ex- DCMU, 50 pM. Rates beside the curves represent pmol tract for 1 min in the dark prior to initiating 02 evolu- 02 evolved. mg chl-’ . h-l. tion with light. 32 Volume 15 1, number 1 FEBS LETTERS January 1983 - a 5 2 6 0 5 0" 4 7.0 7.2 1.4 7.6 7.0 0.0 8.2 PH Fig. 4. Effect of pH on the rate of GOGAT-dependent 02 evolution in a reconstituted chloroplast system. Stan- I I I I I dard assay contained glutamine, 5 mM; 2-oxoglutarate, I 2 3 4 5 2.5 mM and 100~1 of chloroplast extract. The reaction Ferredoxin (uM) mixtures were pre-incubated for 1 min in the dark prior to initiating 02 evolution with light. Fig. 3. Effect of spinach ferredoxin on the rate of GOGAT-dependent 02 evolution in a reconstituted 24- -100 chloroplast system. Assays contained glutamine, 4 mM; , 2-OG 2 mM and 50 ,uI of chloroplast extract. The reac- tion mixtures were pre-incubated for 1 min in the dark prior to initiating 02 evolution with light. 16- achieved in the absence of added ferredoxin when -60 larger volumes of chloroplast extract were em- ployed in the reaction mixture, due to the presence of some ferredoxin in the chloroplast extract which was removed from the pea chloroplasts during the preparation of the reconstitution extracts. GOGAT-dependent 02 evolution in the reconsti- tuted system demonstrated a broad pH optimum with a peak at pH 7.6 (fig. 4). The system also displayed an absolute requirement for Mg’+, which could not be attributed to a requirement of the 0 2 4 6 8 IO 12 14 I'6 photosynthetic electron-transport chain for this M&I, (mM) ion, as demonstrated by the high rate of NADP- Fig. 5. Effect of [MgClz] on the rate of GGGAT- and dependent 02 evolution in the absence of added NADP-dependent 02 evolution in a reconstituted chloro- plast system. Standard GOGAT assay contained gluta- MgCl2 (fig. 5). No GOGAT-dependent 02 evolu- mine, 4 mM; 2-OG, 2 mM and 125 pl of chloroplast tion was observed in the absence of added MgC12. extract. Assay for NADP-dependent 02 evolution was as This requirement for Mg2+ could be replaced to a for GOGAT system, except for chlorophyll, 1OOpg; ferre- certain extent by Ca2+ and Mn2+ but not by K+ or doxin, 125 pg; NADP, 1.0 mM. Reaction mixtures were Na+ (table l), showing the effect to be specific for pre-incubated for 1 min in the dark prior to initiating 02 a divalent cation. evolution with light. 33 Volume 15 1, number 1 FEBSLETTERS January 1983 Table 1 mal rate of 0~ evolution. This did not occur, how- ever, if the GOGAT enzyme was pre-incubated with Effect of various inorganic salts on the rate of GOGAT- dependent 02 evolution in a reconstituted chloroplast 2-OG, prior to the initiation of 02 evolution with system glutamine (trace B, fig. 1) or with light, when both substrates were present (not shown). As this may Salt 02 Evolution indicate some form of allosteric interaction be- (gmol. mg chl-’ . h-‘) tween the GOGAT enzyme and 2-OG, the effect of varying concentrations of this substrate, on the rate MgClz 15.2 of 02 evolution was investigated. While the results 12.3 CaC12 (fig. 6) tend to indicate a normal Michaelis-Menten MnClz 9.9 type relationship between substrate concentration KC1 0.0 NaCl 0.0 and reaction rate, it should be noted that the rates plotted are in fact the steady state rates achieved Standard assay contained glutamine, 4 mM; 2-OG 2 mM, following the lag period, and that this lag period and 150 ~1 of chloroplast extract. The various salts were varied depending on the concentration of 2-OG added to 10 mM after incubation of the reaction mixture present; e.g., 4 min at 0.15 mM 2-OG compared to in the light for 1 min 2 min at 2.0 mM 2-OG.
Load more

Recommended publications
  • Analysis of Functional Domains on Glutamate Synthase
    Turk J Biol 24 (2000) 197–213 © TÜBİTAK Review Article Analysis of Functional Domains on Glutamate Synthase Barbaros NALBANTOĞLU Department of Biochemistry, Faculty of Sciences and Arts, Atatürk University, Erzurum-TURKEY Received: 02.06.1998 Abstract: Glutamate synthases (GOGAT) were analyzed to identify the functional binding domains of the substrate (glutamine) and cofactors (FMN, NAD(P)H, FAD, [3Fe-4S]1+,0 and [4Fe-4S]2+,1+ clusters and ferredoxin) on this enzyme. The published amino acid sequences of six different NAD(P)H- dependent GOGATs (NAD(P)H-GOGAT) and ten different ferredoxin-dependent GOGATs (Fd-GOGAT) were used for this analysis. The amino acid sequences of these sixteen GOGATs were compared with the amino acid sequences of aminotransferases for glutamine, flavoproteins for FMN, flavoproteins and pyridine-nucleotide-dependent enzymes for FAD and NAD(P)H, iron-sulfur proteins for [3Fe- 4S]1+,0 and [4Fe-4S]2+,1+ clusters and ferredoxin-dependent enzymes for ferredoxin. It was determined that Fd-GOGAT has one domain each for glutamine, FMN and [3Fe-4S]1+,0 cluster and two domains each for FAD and ferredoxin; the NADPH-GOGAT α subunit has the same domains as Fd- GOGAT except for the ferredoxin domains, and β subunit has one domain each for NADPH and FAD and two domains for two [4Fe-4S]2+,1+clusters; NADH-GOGAT has the same domains as NADPH- GOGAT. Key Words: Glutamate synthase, glutamine, FMN, FAD, NAD(P)H, iron-sulfur cluster, ferredoxin, binding domain. Glutamat Sentaz Üzerindeki Fonksiyonel Bölgelerin Analizi Özet: Glutamat sentazlar (GOGAT), bu enzim üzerindeki substrat (glutamin) ve kofaktörlerin (FMN, NAD(P)H, FAD, [3Fe-4S]1+,0 ve [4Fe-4S]2+,1+ kümeleri ve ferredoksin) fonksiyonel bağlanma bölgelerini belirlemek için analiz edildi.
    [Show full text]
  • Genomic Deletions Disrupt Nitrogen Metabolism Pathways of a Cyanobacterial Diatom Symbiont
    ARTICLE Received 16 Oct 2012 | Accepted 15 Mar 2013 | Published 23 Apr 2013 DOI: 10.1038/ncomms2748 OPEN Genomic deletions disrupt nitrogen metabolism pathways of a cyanobacterial diatom symbiont Jason A. Hilton1, Rachel A. Foster1,w, H. James Tripp1,w, Brandon J. Carter1, Jonathan P. Zehr1 & Tracy A. Villareal2 Diatoms with symbiotic N2-fixing cyanobacteria are often abundant in the oligotrophic open ocean gyres. The most abundant cyanobacterial symbionts form heterocysts (specialized cells for N2 fixation) and provide nitrogen (N) to their hosts, but their morphology, cellular locations and abundances differ depending on the host. Here we show that the location of the symbiont and its dependency on the host are linked to the evolution of the symbiont genome. The genome of Richelia (found inside the siliceous frustule of Hemiaulus) is reduced and lacks ammonium transporters, nitrate/nitrite reductases and glutamine:2-oxoglutarate aminotransferase. In contrast, the genome of the closely related Calothrix (found outside the frustule of Chaetoceros) is more similar to those of free-living heterocyst-forming cyanobacteria. The genome of Richelia is an example of metabolic streamlining that has implications for the evolution of N2-fixing symbiosis and potentially for manipulating plant–cyanobacterial interactions. 1 Department of Ocean Sciences, University of California, 1156 High Street, Santa Cruz, California 95064, USA. 2 Marine Science Institute, Department of Marine Science, The University of Texas at Austin, 750 Channel View Drive, Port Aransas, Texas 78373, USA. w Present addresses: Department of Biogeochemistry, Max Planck Institute for Marine Microbiology, Celsiusstrasse 1, 28359 Bremen, Germany (R.A.F.); Department of Energy, Joint Genome Institute, 2800 Mitchell Drive, Walnut Creek, California 94598, USA (H.J.T.).
    [Show full text]
  • Light-Independent Nitrogen Assimilation in Plant Leaves: Nitrate Incorporation Into Glutamine, Glutamate, Aspartate, and Asparagine Traced by 15N
    plants Review Light-Independent Nitrogen Assimilation in Plant Leaves: Nitrate Incorporation into Glutamine, Glutamate, Aspartate, and Asparagine Traced by 15N Tadakatsu Yoneyama 1,* and Akira Suzuki 2,* 1 Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan 2 Institut Jean-Pierre Bourgin, Institut national de recherche pour l’agriculture, l’alimentation et l’environnement (INRAE), UMR1318, RD10, F-78026 Versailles, France * Correspondence: [email protected] (T.Y.); [email protected] (A.S.) Received: 3 September 2020; Accepted: 29 September 2020; Published: 2 October 2020 Abstract: Although the nitrate assimilation into amino acids in photosynthetic leaf tissues is active under the light, the studies during 1950s and 1970s in the dark nitrate assimilation provided fragmental and variable activities, and the mechanism of reductant supply to nitrate assimilation in darkness remained unclear. 15N tracing experiments unraveled the assimilatory mechanism of nitrogen from nitrate into amino acids in the light and in darkness by the reactions of nitrate and nitrite reductases, glutamine synthetase, glutamate synthase, aspartate aminotransferase, and asparagine synthetase. Nitrogen assimilation in illuminated leaves and non-photosynthetic roots occurs either in the redundant way or in the specific manner regarding the isoforms of nitrogen assimilatory enzymes in their cellular compartments. The electron supplying systems necessary to the enzymatic reactions share in part a similar electron donor system at the expense of carbohydrates in both leaves and roots, but also distinct reducing systems regarding the reactions of Fd-nitrite reductase and Fd-glutamate synthase in the photosynthetic and non-photosynthetic organs.
    [Show full text]
  • Lake Superior Phototrophic Picoplankton: Nitrate Assimilation
    LAKE SUPERIOR PHOTOTROPHIC PICOPLANKTON: NITRATE ASSIMILATION MEASURED WITH A CYANOBACTERIAL NITRATE-RESPONSIVE BIOREPORTER AND GENETIC DIVERSITY OF THE NATURAL COMMUNITY Natalia Valeryevna Ivanikova A Dissertation Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY May 2006 Committee: George S. Bullerjahn, Advisor Robert M. McKay Scott O. Rogers Paul F. Morris Robert K. Vincent Graduate College representative ii ABSTRACT George S. Bullerjahn, Advisor Cyanobacteria of the picoplankton size range (picocyanobacteria) Synechococcus and Prochlorococcus contribute significantly to total phytoplankton biomass and primary production in marine and freshwater oligotrophic environments. Despite their importance, little is known about the biodiversity and physiology of freshwater picocyanobacteria. Lake Superior is an ultra- oligotrophic system with light and temperature conditions unfavorable for photosynthesis. Synechococcus-like picocyanobacteria are an important component of phytoplankton in Lake Superior. The concentration of nitrate, the major form of combined nitrogen in the lake, has been increasing continuously in these waters over the last 100 years, while other nutrients remained largely unchanged. Decreased biological demand for nitrate caused by low availabilities of phosphorus and iron, as well as low light and temperature was hypothesized to be one of the reasons for the nitrate build-up. One way to get insight into the microbiological processes that contribute to the accumulation of nitrate in this ecosystem is to employ a cyanobacterial bioreporter capable of assessing the nitrate assimilation capacity of phytoplankton. In this study, a nitrate-responsive biorepoter AND100 was constructed by fusing the promoter of the Synechocystis PCC 6803 nitrate responsive gene nirA, encoding nitrite reductase to the Vibrio fischeri luxAB genes, which encode the bacterial luciferase, and genetically transforming the resulting construct into Synechocystis.
    [Show full text]
  • The Pleiotropic Effects of the Glutamate Dehydrogenase (GDH) Pathway In
    Mara et al. Microb Cell Fact (2018) 17:170 https://doi.org/10.1186/s12934-018-1018-4 Microbial Cell Factories REVIEW Open Access The pleiotropic efects of the glutamate dehydrogenase (GDH) pathway in Saccharomyces cerevisiae P. Mara1,4* , G. S. Fragiadakis3, F. Gkountromichos2,5 and D. Alexandraki2,3 Abstract Ammonium assimilation is linked to fundamental cellular processes that include the synthesis of non-essential amino acids like glutamate and glutamine. In Saccharomyces cerevisiae glutamate can be synthesized from α-ketoglutarate and ammonium through the action of NADP-dependent glutamate dehydrogenases Gdh1 and Gdh3. Gdh1 and Gdh3 are evolutionarily adapted isoforms and cover the anabolic role of the GDH-pathway. Here, we review the role and function of the GDH pathway in glutamate metabolism and we discuss the additional contributions of the pathway in chromatin regulation, nitrogen catabolite repression, ROS-mediated apoptosis, iron defciency and sphingolipid-dependent actin cytoskeleton modulation in S.cerevisiae. The pleiotropic efects of GDH pathway in yeast biology highlight the importance of glutamate homeostasis in vital cellular processes and reveal new features for conserved enzymes that were primarily characterized for their metabolic capacity. These newly described features constitute insights that can be utilized for challenges regarding genetic engineering of glutamate homeostasis and maintenance of redox balances, biosynthesis of important metabolites and production of organic substrates. We also conclude that the discussed pleiotropic features intersect with basic metabolism and set a new background for further glutamate-dependent applied research of biotechnological interest. Keywords: Glutamate dehydrogenase, GDH1, GDH2, GDH3, Ammonium assimilation, GABA shunt, ROS-mediated apoptosis, Chromatin regulation, Nitrogen catabolite repression, S.
    [Show full text]
  • Trichodesmium Spp., the Gldglu Ratio Closely Approximated the Glnlakg Ratio Over the Die1 Cycle
    MARINE ECOLOGY PROGRESS SERIES Published November 3 Mar Ecol Prog Ser Nitrogen fixation, uptake and metabolism in natural and cultured populations of Trichodesmium spp. Margaret R. Mulholland*, Douglas G. Capone*" Chesapeake Biological Laboratory, University of Maryland Center for Environmental Science, PO Box 38. Solomons, Maryland 20688, USA ABSTRACT: Uptake rates of several combined N sources, NZfixation, intracellular glutamate (glu) and glutamine (gln) pools, and glutamine synthetase (GS) activity were measured in natural populations and a culture of Trichodesmium IMSlOl grown on seawater medium without added N. In cultured populations, the ratio of GS transferase/biosynthetic activity (an index of the proportion of the GS pool that is active) was lower, and intracellular pools of glu and gln and the ratios of gldglu and glnla- ketoglutarate (glnlakg) ratios were higher when NZ fixation was highest (mid-day). There was an excess capacity for NH,' assimilation via GS, indicating that this was not the rate-limiting step in N uti- lization. In natural populations of Trichodesmium spp., the gldglu ratio closely approximated the glnlakg ratio over the die1 cycle. High gln/glu and gln/akg ratios were noted in near-surface popula- tion~.These ratios decreased in samples collected from greater depths. Natural populations of Tn- chodesmium spp. showed a high capacity for the uptake of NH4+,glu, and mixed amino acids (AA). Rates of NO3- and urea uptake were low. NH,+ accumulated in the culture medium during growth and rates of NH4+ uptake showed a positive relationship with the NH4+ concentration in the medium. Although rates of NZfixation were highest and accounted for the majority of the total measured N uti- lization during mid-day, rates of NH4+uptake exceeded rates of NZfixation throughout much of the die1 cycle.
    [Show full text]
  • Compartmentalized Cyanophycin Metabolism in the Diazotrophic Filaments of a Heterocyst- Forming Cyanobacterium
    Compartmentalized cyanophycin metabolism in the diazotrophic filaments of a heterocyst- forming cyanobacterium Mireia Burnat, Antonia Herrero, and Enrique Flores1 Instituto de Bioquímica Vegetal y Fotosíntesis, Consejo Superior de Investigaciones Científicas and Universidad de Sevilla, E-41092 Seville, Spain Edited by Robert Haselkorn, University of Chicago, Chicago, IL, and approved January 24, 2014 (received for review October 2, 2013) Heterocyst-forming cyanobacteria are multicellular organisms in cyanobacterial filament (4, 6). Indeed, when incubated under which growth requires the activity of two metabolically interde- proper conditions, isolated heterocysts can produce glutamine pendent cell types, the vegetative cells that perform oxygenic that is released to the surrounding medium (6). In addition to photosynthesis and the dinitrogen-fixing heterocysts. Vegetative glutamate, two compounds that can be transferred from vege- cells provide the heterocysts with reduced carbon, and heterocysts tative cells to heterocysts are alanine and sucrose (8). Alanine provide the vegetative cells with fixed nitrogen. Heterocysts con- can be an immediate source of reducing power in the heterocyst, spicuously accumulate polar granules made of cyanophycin [multi- where it is metabolized by catabolic alanine dehydrogenase, the L-arginyl-poly (L-aspartic acid)], which is synthesized by cyano- product of ald (9). Sucrose, a universal vehicle of reduced carbon phycin synthetase and degraded by the concerted action of in plants, appears to have a similar role within the diazotrophic cyanophycinase (that releases β-aspartyl-arginine) and isoaspartyl cyanobacterial filament, because diazotrophic growth requires dipeptidase (that produces aspartate and arginine). Cyanophycin that sucrose is produced in vegetative cells and hydrolyzed by a synthetase and cyanophycinase are present at high levels in the specific invertase, InvB, in the heterocysts (10–13).
    [Show full text]
  • Glutamate Synthase: Structural, Mechanistic and Regulatory Properties, and Role in the Amino Acid Metabolism
    Photosynthesis Research (2005) 83: 191–217 Ó Springer 2005 Review Glutamate synthase: structural, mechanistic and regulatory properties, and role in the amino acid metabolism Akira Suzuki1,* & David B. Knaff2 1Unite´ de Nutrition Azote´e des Plantes, Institut National de la Recherche Agronomique, Route de Saint-Cyr, 78026 Versailles cedex, France; 2Department of Chemistry and Biochemistry, Texas Tech University, P.O. Box 41061, Lubbock, TX 79409-1061, USA; *Author for correspondence (e-mail: [email protected]; fax: +33-1-30833096) Received 28 June 2004; accepted in revised form 20 September 2004 Key words: ammonium assimilation, glutamate synthase, glutamine synthetase, higher plants, nitrogen metabolism Abstract Ammonium ion assimilation constitutes a central metabolic pathway in many organisms, and glutamate synthase, in concert with glutamine synthetase (GS, EC 6.3.1.2), plays the primary role of ammonium ion incorporation into glutamine and glutamate. Glutamate synthase occurs in three forms that can be dis- tinguished based on whether they use NADPH (NADPH-GOGAT, EC 1.4.1.13), NADH (NADH-GO- GAT, EC 1.4.1.14) or reduced ferredoxin (Fd-GOGAT, EC 1.4.7.1) as the electron donor for the (two- electron) conversion of L-glutamine plus 2-oxoglutarate to L-glutamate. The distribution of these three forms of glutamate synthase in different tissues is quite specific to the organism in question. Gene structures have been determined for Fd-, NADH- and NADPH-dependent glutamate synthases from different organisms, as shown by searches in nucleic acid sequence data banks. Fd-glutamate synthase contains two electron-carrying prosthetic groups, the redox properties of which are discussed.
    [Show full text]
  • Dicarboxylate Transport (Photorespiration/Malate Shuttle/Glutamine Transport) S
    Proc. NatL Acad. Sci. USA Vol. 80, pp. 1290-1294, March 1983 Botany An Arabidopsis thaliana mutant defective in chloroplast dicarboxylate transport (photorespiration/malate shuttle/glutamine transport) S. C. SOMERVILLE*t AND W. L. OGREN*t *Department of Agronomy, University of Illinois, Urbana, Illinois 61801; and tU. S. Department of Agriculture, Agricultural Research Service, Urbana, Illinois 61801 Communicated by Harry Beevers, November 8, 1982 ABSTRACT Reactions of the photorespiratory pathway of C3 ically to CO2 at rates that significantly reduce net CO2 assim- plants are found in three subcellular organelles. Transport pro- ilation (9), and ammoniaaccumulates totoxiclevels (8). Because cesses are, therefore, particularly important for maintaining the glutamate is synthesized in the chloroplast (7) and consumed uninterrupted flow of carbon through this pathway. We describe in the peroxisome, the transfer ofglutamate and 2-oxoglutarate here the isolation and characterization of a photorespiratory mu- between these two organelles is a necessary component ofpho- tant of Arabidopsis thaliana defective in chloroplast dicarboxylate torespiratory nitrogen metabolism. transport. Genetic analysis indicates the defect is due to a simple, Transport systems operating at the peroxisome-bounding recessive, nuclear mutation. Glutamine and inorganic phosphate been characterized. However, studies with transport are unaffected by the mutation. Thus, in contrast to pre- membrane have not vious reports for pea and spinach, glutamine uptake by Arabi- isolated chloroplasts have revealed the presence of a trans- dopsis chloroplasts is mediated by a transporter distinct from the porter, designated the dicarboxylate transporter, which cata- dicarboxylate transporter. Both the inviability and the disruption lyzes the counter-exchange of several dicarboxylic acids across of amino-group metabolism of the mutant under photorespiratory the chloroplast inner membrane (10).
    [Show full text]
  • Plastid-Localized Amino Acid Biosynthetic Pathways of Plantae Are Predominantly Composed of Non-Cyanobacterial Enzymes
    Plastid-localized amino acid biosynthetic pathways of Plantae are predominantly SUBJECT AREAS: MOLECULAR EVOLUTION composed of non-cyanobacterial PHYLOGENETICS PLANT EVOLUTION enzymes PHYLOGENY Adrian Reyes-Prieto1* & Ahmed Moustafa2* Received 1 26 September 2012 Canadian Institute for Advanced Research and Department of Biology, University of New Brunswick, Fredericton, Canada, 2Department of Biology and Biotechnology Graduate Program, American University in Cairo, Egypt. Accepted 27 November 2012 Studies of photosynthetic eukaryotes have revealed that the evolution of plastids from cyanobacteria Published involved the recruitment of non-cyanobacterial proteins. Our phylogenetic survey of .100 Arabidopsis 11 December 2012 nuclear-encoded plastid enzymes involved in amino acid biosynthesis identified only 21 unambiguous cyanobacterial-derived proteins. Some of the several non-cyanobacterial plastid enzymes have a shared phylogenetic origin in the three Plantae lineages. We hypothesize that during the evolution of plastids some enzymes encoded in the host nuclear genome were mistargeted into the plastid. Then, the activity of those Correspondence and foreign enzymes was sustained by both the plastid metabolites and interactions with the native requests for materials cyanobacterial enzymes. Some of the novel enzymatic activities were favored by selective compartmentation should be addressed to of additional complementary enzymes. The mosaic phylogenetic composition of the plastid amino acid A.R.-P. ([email protected]) biosynthetic pathways and the reduced number of plastid-encoded proteins of non-cyanobacterial origin suggest that enzyme recruitment underlies the recompartmentation of metabolic routes during the evolution of plastids. * Equal contribution made by these authors. rimary plastids of plants and algae are the evolutionary outcome of an endosymbiotic association between eukaryotes and cyanobacteria1.
    [Show full text]
  • Evidence for Ferredoxin Glutamate Synthase Activity in Grape Shoot
    Vitis 36 (4), 207-208 (1997) Research Note and 14 mM ß-mercapto-ethanol, ß-ME). After centrifuga­ tion, proteins were precipitated with 5 volumes of 0.1 M ammoniumacetatein methanol at -20 oc ovemight, washed Evidence for ferredoxin glutamate synthase twice with fresh 0.1 M ammoniumacetatein methanol, then activity in grape shoot tissues twice with -20 oc acetone. The proteins were resuspended in sample buffer, their protein concentration determined (BRADFORD 1976), then loaded onto SDS mini gels (Mini-PROTEAN II apparatus, Bio-Rad Laboratories, Richmond, CA). Proteins were trans­ ferred to a nitrocellulose membrane using a Bio-Rad Mini Summary : Ferredoxin glutamate synthase (ferredoxin Trans-Blot Electrophoretic Transfer Cell. Membranes were glutamine: oxoglutarate aminotransferase, or Fd-GOGAT, probed with lgG anti-rice Fd-GOGAT (SuzuKr et al. 1982) EC 1.4. 7 .I) activity was detected in grapevine ( Vitis vinifera L. ). and developed using a Bio-Rad Immun-Blot Assay Kit Highest Fd-GOGAT activity was found in Iamina tissue. Moder­ (AP goat anti-rabbit IgG). ate Ievels of activity were found in petiole, tendril, rachis, and For the enzyme assay, frozen tissues were ground with flower tissues. No activity was detected in extracts from pedicel 75% (w/w) PVPP, then added to 12 volumes 200 mM phos­ tissue. Western blotting with rice Fd-GOGAT antibodies resulted phate buffer, pH 7 .6, containing 2.5 mM EDTA, 75 mM borax in a positive reaction with single grape proteinband of73 kDa in decahydrate, 28 mM ß-ME, 5 mM dithiothreitol (DTT), all tissue types tested. 0.2 mM PMSF, and 75 % (w/w) PVPP.
    [Show full text]
  • Allelic Variants of Glutamine Synthetase and Glutamate Synthase Genes in a Collection of Durum Wheat and Association with Grain Protein Content
    diversity Article Allelic Variants of Glutamine Synthetase and Glutamate Synthase Genes in a Collection of Durum Wheat and Association with Grain Protein Content Domenica Nigro 1, Stefania Fortunato 1, Stefania Lucia Giove 2, Giacomo Mangini 1, Ines Yacoubi 3, Rosanna Simeone 1, Antonio Blanco 1 and Agata Gadaleta 2,* 1 Department of Soil, Plant & Food Sciences, Plant Breeding Section, University of Bari Aldo Moro, Via Amendola, 165/a, 70126 Bari BA, Italy; [email protected] (D.N.); [email protected] (S.F.); [email protected] (G.M.); [email protected] (R.S.); [email protected] (A.B.) 2 Department of Agricultural & Environmental Science, University of Bari Aldo Moro, Via Amendola, 165/a, 70126 Bari BA, Italy; [email protected] 3 Biotechnology and plant improvement laboratory, Biotechnology Centre of Sfax Tunisia, Kef 7119, Tunisia; [email protected] * Correspondence: [email protected] Received: 6 October 2017; Accepted: 7 November 2017; Published: 16 November 2017 Abstract: Wheat is one of the most important crops grown worldwide. Despite the fact that it accounts for only 5% of the global wheat production, durum wheat (Triticum turgidum L. subsp. durum) is a commercially important tetraploid wheat species, which originated and diversified in the Mediterranean basin. In this work, the candidate gene approach has been applied in a collection of durum wheat genotypes; allelic variants of genes glutamine synthetase (GS2) and glutamate synthase (GOGAT) were screened and correlated with grain protein content (GPC). Natural populations and collections of germplasms are quite suitable for this approach, as molecular polymorphisms close to a locus with evident phenotypic effects may be closely associated with their character, providing a better physical resolution than genetic mapping using ad hoc constituted populations.
    [Show full text]