The intracellular location of the enzymes of nitrate assimilation in the apices of seedling pea roots

The intracellular distribution of the enzymes of nitrate and ammonia assimilation in apical cells of pea (Pisum sativum L.) roots is described. Nitrate reductase (EC 1.6.6.2) was found to have no organelle association, and is considered to be located in the cytosol or possibly loosely bound to the outside of an organelle. Nitrite reductase and glutamate synthase (EC 2.6.1.53) are plastid located, as is glutamine synthetase (EC 6.3.1.2) although this enzyme also has activity in the cytosol. Glutamate dehydrogenase (EC 1.4.1.3) was found only in the mitochondrion.     

Nitrogen nutrition and the development of biochemical functions associated with nitrogen fixation and ammonia assimilation of nodules on cowpea seedlings

During early development (up to 18 d after sowing) of nodules of an effective cowpea symbiosis (Vigna unguiculata (L.) Walp cv. Vita 3: Rhizobium strain CB756), rapidly increasing nitrogenase (EC 1.7.99.2) activity and leghaemoglobin content were accompanied by rapid increases in activities of glutamine synthetase (EC 6.3.1.2), glutamate synthase (EC 2.6.1.53), enzymes of denovo purine synthesis (forming inosine monophosphate) xanthine oxidoreductase (EC 1.2.3.2), urate oxidase (EC 1.7.3.3), phosphoenolpyruvate carboxylase (EC 4.1.1.31) and led to increased export of ureides (allantoin and allantoic acid) to the shoot of the host plant in the xylem. Culturing plants with the nodulated root systems maintained in the absence of N₂ (in 80 Ar: 20 O₂, v/v) had little effect on the rates of induction and increase in nitrogenase activity and leghaemoglobin content but, in the absence of N₂ fixation and consequent ammonia production by bacteroids, there was no stimulation of activity of enzymes of ammonia assimilation or of the synthesis of purines or ureides. Addition of NO ₃ ^- (0.1–0.2 mM) relieved host-plant nitrogen deficiency caused by the Ar: O₂ treatment but failed to increase levels of enzymes of N metabolism in either the bacteroid or the plant-cell fractions of the nodule. Premature senescence in Ar: O₂-grown nodules occurred at 18–20 d after sowing, and resulted in reduced levels of nitrogenase activity and leghaemoglobin but increased the activity of hydroxybutyrate oxidoreductase (EC 1.1.1.30).     

Primary metabolism in N2-fixing Alnus incana-Frankia symbiotic root nodules studied with 15N and 31P nuclear magnetic resonance spectroscopy

The primary nitrogen metabolism of the N₂-fixing root nodule symbiosis Alnus incana (L.)–Frankia was investigated by ³¹P and ¹⁵N nuclear magnetic resonance (NMR) spectroscopy. Perfusion of root nodules in a pulse–chase approach with ¹⁵N- or ¹⁴N-labeled NH₄⁺ revealed the presence of the amino acids alanine (Ala), -amino butyric acid, glutamine (Gln), glutamic acid (Glu), citrulline (Cit) and arginine (Arg). Labeling kinetics of the Gln amide-N and -amino acids suggested that the glutamine synthetase (GS; EC 6.3.1.2)–glutamate synthase (GOGAT; EC 1.4.1.13) pathway was active. Inhibition of the GS-catalyzed reaction by methionine sulphoximine abolished incorporation of ¹⁵N. Cit was labeled in all three N positions but most rapidly in the position, consistent with carbamoyl phosphate as the precursor to which Gln could be the amino donor catalyzed by carbamoyl phosphate synthase (CPS; EC 6.3.5.5). Ala biosynthesis occurred consistent with a flux of N in the sequence Gln–Glu–Ala. ³¹P NMR spectroscopy in vivo and of extracts revealed several metabolites and was used in connection with the ¹⁵N pulse–chase experiment to assess general metabolic status. Stable concentrations of ATP and UDP-glucose during extended perfusions showed that the overall root nodule metabolism appeared undisturbed throughout the experiments. The metabolic pathways suggested by the NMR results were confirmed by high activities of the enzymes GS, NADH-GOGAT and ornithine carbamoyltransferase (OCT; EC 2.1.3.3). We conclude that the primary pathway of NH₄⁺ assimilation in A. incana root nodules occurs through the GS–GOGAT pathway. Biosynthesis of Cit through GS–CPS–OCT is important and is a link between the first amino acid Gln and this final transport and storage form of nitrogen.Abbreviations AlaDH l-Alanine dehydrogenase - Cit Citrulline - CPS Carbamoyl phosphate synthase - GABA -Amino butyric acid - GOGAT Glutamate synthase - GS Glutamine synthetase - MDH Malate dehydrogenase - MSO Methionine sulphoximine - NMR Nuclear magnetic resonance - OCT Ornithine carbamoyltransferase - PEPC Phosphoenolpyruvate decarboxylase - UDPGlc Uridine 5-diphosphoglucose     

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     

ent-Kaurene biosynthesis in a cell-free system from wheat (Triticum aestivum L.) seedlings and the localisation of ent-kaurene synthetase in plastids of three species

A cell-free system capable of converting [¹⁴C]geranylgeranyl diphosphate to ent-[¹⁴C]kaurene and to an unidentified acid-hydrolysable compound was obtained from the basal portions of 5-d-old shoots of wheat seedlings (Triticum aestivum L.). By means of marker enzyme activities, the synthesis of ent-kaurene and the unknown compound could be quantitatively assigned to a plastid fraction obtained by Percoll-gradient centrifugation of the homogenate. The enzyme activities were located within the plastids, probably in the stroma, because they withstood trypsin treatment of the intact plastids, and the plastids had to be broken to release the activity, which was then obtained in soluble form. Plastid membranes had no activity. Plastid stroma preparations obtained from pea (Pisum sativum L.) shoot tips and pumpkin (Cucurbita maxima L.) endosperm also yielded ent-kaurene synthetase activity, but did not form the unknown compound. The exact nature of the active plastids was not ascertained, but the use of methods for proplastid isolation was essential for full activity, and the active tissues are all known to contain high proportions of proplastids, developing chloroplasts or leucoplasts. We therefore believe that ent-kaurene synthesis may be limited to these categories. Mature chloroplasts from the wheat leaves did not contain ent-kaurene synthetase activity and did not yield the unknown component. Incorporation of [¹⁴C]geranylgeranyl diphosphate into ent-[¹⁴C]kaurene and the unknown component was assayed by high-performance liquid chromatography with on-line radiocounting. ent-[¹⁴C]Kaurene was identified by Kovats retention index and full mass spectra obtained by combined gas chromatography-mass spectrometry. The unknown component was first believed to be copalyl diphosphate, because it yielded a compound on acid hydrolysis, which migrated like copalol on high-performance liquid chromatography and gave a mass spectrum very similar to that of authentic copalol. However, differences in the mass spectrum and in retention time on capillary gas chromatography excluded identity with copalol. Furthermore, the unhydrolysed compound was not converted to ent-kaurene by a cell-free system from C. maxima endosperm as copalyl diphosphate would have been.Abbreviations ADH alcohol dehydrogenase - AMO 1618 2isopropyl-4-(trimethylammoniumchloride)-5-methylphenyl piperi-dine-1-carboxylate - BSA bovine serum albumin - DTT dithioth-reitol - GA_n gibberellin A_n - GAPDH NADP⁺-glyceraldehyde 3-phosphate dehydrogenase - GC-MS combined gas chromatography-mass spectrometry - GGPP all trans-isomer of geranyl-geranyl diphosphate - KS ent-kaurene synthetase - MDH malate dehydrogenase - MAA mevalonate activating activity - SOR shikimate oxidoreductase We thank Mrs. Gudrun Bodtke and Mrs. Dorothee Dasbach for able technical assistance, Prof. L.N. Mander (Australian National University, Canberra, Australia) for ent-[²H₂]kaurene and Dr. Yuji Kamiya (RIKEN, Saitama, Japan) for geranylgeraniol and copalol. The work was supported by the Deutsche Forschungsgemeinschaft.     

Electron transport in purified glyoxysomal membranes from castor-bean endosperm

Glyoxysomes isolated from castor-bean (Ricinus communis L.) endosperm were treated with water, 0.2 M KCl, 1 M KCl, or 0.1 M Na₂CO₃. Glyoxysomal sacs, i.e. membranes which retained some visible matrix, resulted from the treatments with water and KCl. Glyoxysomal ghosts, i.e. intact membranes free of matrix, were only obtained following treatment with carbonate. The ghosts were free of activities of matrix enzymes, particularly palmitoyl-CoA oxidation, isocitrate dehydrogenase (EC 1.1.1.42) and isocitrate lyase (EC 4.1.3.1), and contained only negligible amounts of malate synthase (EC 4.1.3.2), malate dehydrogenase (EC 1.1.1.37), -hydroxyacyl-CoA dehydrogenase (EC 1.1.1.98) and catalase (EC 1.11.1.6). Distribution and appearance of membrane-associated particles in the protoplasmic and ectoplasmic faces of freeze-fracture replicas of the glyoxysomal membrane were the same in intact tissue, isolated glyoxysomes, and ghosts. Membranes purified by treatment with 0.2 M KCl or 0.1 M carbonate catalyzed the reduction of cytochrome-c when NADH or NADPH was provided as the electron donor. -Oxidation, localized in the matrix, could be linked to reduction of cytochrome-c or ferricyanide when purified membranes were combined with the matrix supernatant. Cytochrome-c could also be reduced by coupling enzyme activities in the matrix, NADP-isocitrate dehydrogenase or malate dehydrogenase, with those of the membrane. These results indicate that electrons from -oxidation, malate oxidation or isocitrate oxidation can be transferred directly to the redox components of the glyoxysomal membrane. We, therefore, conclude that any NADH and NADPH formed by enzymes in the matrix can be recycled continuously within the organelle.Abbreviations EF ectoplasmic face - ER endoplasmic reticulum - PF protoplasmic face     

Appearance of nitrite reductase in cotyledons of the mustard (Sinapis alba L.) seedling as affected by nitrate,phytochrome and photooxidative damage of plastids

Nitrite reductase (NIR; EC 1.7.7.1) is a central enzyme in nitrate assimilation and is localized in plastids. The present study concerns the regulation of the appearance of NIR in cotyledons of the mustard (Sinapis alba L.) seedling. It was shown that light exerts its positive control over the nitrate-mediated induction of NIR via the farred-absorbing form of phytochrome. Without nitrate the light effect cannot express itself; even though the light signal is accumulated in the cotyledons it remains totally cryptic in the absence of nitrate. Moreover, it was recognised that intact plastids are important in the control of the appearance of NIR. If the plastids are damaged by photooxidation the action of nitrate and phytochrome on NIR appearance is abolished. The appearance of nitrate reductase (NR; EC 1.6.6.1) responds similarly to photooxidative damage even though this enzyme is cytosolic. While the data strongly indicate that some plastidic signal is a prerequisite for the nitrate-induced and phytochrome-modulated appearance of NIR and NR, the possibility could not be ruled out that photooxidative damage affects the accumulation of NIR in the organelle.Abbreviations c continuous - D darkness - FR far-red light - NADP-GPD NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.1.13) - NF Norflurazon - NIR nitrite reductase (EC 1.7.7.1.) - NR nitrate reductase (EC 1.6.6.1) - Pfr phytochrome (far-red light obtained with RG9 glass filter - R red light - RG9-light long wavelenght far-red light obtained with RG9 glass filter - RuBPCase ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) - WL white light - WL_s strong white light (28 W m^-2)     

Drought Stress Response on Some Key Enzymes of Cowpea (<Emphasis Type="Italic">Vigna unguiculata</Emphasis> L. Walp.) Nodule Metabolism

A greenhouse experiment was carried out aiming to evaluate the response to drought stress of cowpea nodule enzymatic activities during different plant developmental stages leading to biological N₂ fixation. Stress was applied by controlling soil’s water-potential through a porous cup. Cowpea plants cv IPA 205 were grown in pots with yellow latosol soil under three different matric potential (ψ_m) treatments. Even with high evaporative demand and limited soil water availability, cowpea could not induce an extremely low leaf water potential (ψ_w). Sap ureides concentration in cowpea declined during the drought stress period. There was a decline in enzyme activity in the metabolic pathways concerned with N₂ fixation: NADH-dependent glutamate synthase (EC 1.4.1.14), glutamine synthetase (EC 6.3.1.2) and phosphoenolpyruvate carboxylase (EC 4.1.1.31). In contrast, an increase in glutamate dehydrogenase (EC 1.4.1.4) was observed as the ψ_m declined. Metabolism associated with N₂ assimilation was impaired every time that the ψ_w was reduced below −0.73 MPa as had happened in the stressed treatments. The stress applied by the porous cup was gradual and the plant recovered its turgor, avoiding permanent deleterious alterations in the cellular metabolism, even from a limited cowpea-growth ψ_m.     

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.     

The localization of some coenzyme A-dependant enzymes in rat liver mitochondria

1. CoA, acetyl-CoA, l-carnitine and acetyl-l-carnitine when added to rat liver mitochondria equilibrate with approximately two-thirds of the total intramitochondrial water. The mitochondrial space calculated to be freely permeable to these solutes was identical with that obtained for sucrose. 2. Acetyl-CoA is rapidly deacylated by rat liver mitochondria at 0 degrees C, and special precautions are required to measure its mitochondrial permeation. 3. Rat liver mitochondria were separated into fractions that correspond to the inner membrane, the outer membrane, and the soluble proteins of the matrix and intermembrane compartment. Soluble enzymes considered to be located in the matrix were citrate synthase (EC 4.1.3.7), palmitoyl-CoA dehydrogenase (EC 1.3.2.2), electron-transferring flavoprotein, medium-chain-length ATP-specific fatty acyl-CoA synthetase (EC 6.2.1.2), l-3-hydroxybutyryl-CoA dehydrogenase (EC 1.1.1.35) and 3-keto-acyl-CoA thiolase (EC 2.3.1.16). Carnitine palmitoyltransferase (EC 2.3.1.-) is largely associated with the inner-membrane fraction. A long-chain-length ATP-specific fatty acyl-CoA synthetase (EC 6.2.1.3) is associated with the outer-membrane fraction.     

Assimilation of exogenous and dinitrogen-derived13NH 4 + byAnabaena azollae separated fromAzolla caroliniana Willd

Anabaena azollae was isolated fromAzolla caroliniana by the gentle roller method and differential centrifugation. Incubation of suchAnabaena preparations for 10 min with [¹³N]N₂ resulted in the formation of four radioactive compounds; ammonium, glutamine, glutamate and alanine. Ammonium accounted for 66% of the total radioactivity recovered and 58% of the ammonium was in an extracellular fraction. Since essentially no extracellular¹³N-labeled organic compounds were found, it appears that ammonium is the compound most probably made available toAzolla during dinitrogen-dependent growth of the association.The kinetics of incorporation of exogenous¹³NH ₄ ⁺ into glutamine and glutamate were characteristic of a precursor (glutamine)-product (glutamate) relationship and consistent with assimilation by the glutamine synthetase-glutamate synthase pathway. The results of experiments using the glutamine synthetase inhibitor, methionine sulfoximine, the glutamate synthase inhibitor, diazo-oxonorleucine, and increasing the ammonium concentration to greater than 1 mM, provided evidence for assimilation primarily by the glutamine synthetase-glutamate synthase pathway with little or no contribution from biosynthetic glutamate dehydrogenase.While showing that N₂ fixation and NH ₄ ⁺ assimilation were not tightly coupled metabolic processes in symbioticAnabaena, these results reflect a composite picture and do not indicate the extent to which ammonium assimilatory enzymes might be regulated in filaments associated with specific stages in theAzolla-Anabaena developmental profile.Non-standard abbreviations DON 6-Diazo-5-oxo-l-norleucine - GDH glutamate dehydrogenase - GOGAT glutamate synthase - GS glutamine synthetase - MSX l-methionine-Dl-sulfoximine     

Ammonium assimilation in an Arthrobacter sp. “fluorescens”

Ammonium assimilation was studied in a nitrogenfixing Arthrobacter strain grown in both batch and ammonium-limited continuous cultures. Arthrobacter sp. fluorescens grown in nitrogen-free medium or at low ammonium levels assimilated NH ₄ ⁺ via the glutamine synthetase/glutamate synthase pathway. When ammonium was in excess it was assimilated via the alanine dehydrogenase pathway. Very low levels of glutamate dehydrogenase were found, irrespective of growth conditions.Abbreviations GS glutamine synthetase - GOGAT glutamine oxoglutarate aminotransferase - GDH glutamate dehydrogenase - ADH alanine dehydrogenase - GOT glutamate oxaloacetate transaminase - GPT glutamate pyruvate transaminase     

Localization of enzymes of assimilatory sulfate reduction in pea roots

The localization of enzymes of assimilatory sulfate reduction was examined in roots of 5-d-old pea (Pisum sativum L.) seedlings. During an 8-h period, roots of intact plants incorporated more label from ³⁵SO ₄ ^2- in the nutrient solution into the amino-acid and protein fractions than shoots. Excised roots and roots of intact plants assimilated comparable amounts of radioactivity from ³⁵SO ₄ ^2- into the amino-acid and protein fractions during a 1-h period, demonstrating that roots of pea seedlings at this stage of development were not completely dependent on the shoots for reduced sulfur compounds. Indeed, these roots contained activities of ATP-sulfurylase (EC 2.7.7.4), adenosine 5-phosphosulfate sulfotransferase, sulfite reductase (EC 1.8.7.1) and O-acetyl-l-serine sulfhydrylase (EC 4.2.99.8) at levels of 50, 30, 120 and 100%, respectively, of that in shoots. Most of the extractable activity of adenosine 5-phosphosulfate sulfotransferase was detected in the first centimeter of the root tip. Using sucrose density gradients for organelle separation from this part of the root showed that almost 40% of the activity of ATP-sulfurylase, adenosine 5-phosphosulfate sulfotransferase and sulfite reductase banded with the marker enzyme for proplastids, whereas only approximately 7% of O-acetyl-l-serine sulfhydrylase activity was detected in these fractions. Because their distributions on the gradients were very similar to that of nitrite reductase, a proplastid enzyme, it is concluded that ATP-sulfurylase, adenosine 5-phosphosulfate sulfotransferase and sulfite reductase are also exclusively or almost exclusively localized in the proplastids of pea roots. O-Acetyl-l-serine sulfhydrylase is predominantly present in the cytoplasm.Abbreviation APSSTase adenosine 5-phosphosulfate sulfotransferase     

Expression of glutamine synthetase during the anaerobic germination of Oryza sativa L.

Glutamine synthetase (GS; EC.6.3.1.2.) occurs as cytosolic (GS₁) and plastidic (GS₂) polypeptides. This paper describes the expression of GS isoenzymes in coleoptile during the anaerobic germination of rice (Oryza sativa L.) and the influence of exogenous nitrate on this. By immunoprecipitation with anti-GS serum, two polypeptides of 41- and 44-kDa were detected of which the former was predominant. After fractionation by ion-exchange chromatography, the 41 and 44 kDa bands were identified as GS₁ and GS₂, respectively. Northern blot analysis with specific probes showed the presence of mRNA for cytosolic GS but not for the plastidic form. The presence of exogenous nitrate did not alter the activity and expression of GS in the coleoptile. The role of GS during the anaerobic germination of rice seems to induce the re-assimilation of ammonia rather than the assimilation of nitrate.Abbreviations GS glutamine synthetase - GS₁ cytosolic glutamine synthetase - GS₂ platidic glutamine synthetase We are grateful to Dr. Julie V. Cullimore for providing GS anti-serum and clones. The research was supported by the National Research Council of Italy, special project RAISA, sub-project N. 2 paper N. 1586.     

Immunocytochemical Localization of Glutamine Synthetase in Organs of Phaseolus vulgaris L

Glutamine synthetase was localized in nodules, roots, stems, and leaves of red kidney bean (Phaseolus vulgaris L.) by immunocytochemistry. Affinity purified antibodies reactive with glutamine synthetase were prepared using purified nodule-enhanced glutamine synthetase. Immunogold labeling was observed in the cell cytoplasm in each plant organ. In nodules, the labeling was more intense in the infected cells than in the uninfected cells. No labeling was observed in nodule bacteroids, peribacteroid spaces, or in peribacteroid membranes, while previous reports of glutamine synthetase immunolabeling of legume nodules showed labeling in the bacteroid fraction. Significant labeling was observed in nodule proplastids which contained starch granules. Substantial labeling was also observed in leaf chloroplasts. No labeling was observed in other organelles including mitochondria, peroxisomes, and endoplasmic reticulum. Preimmune IgGs did not bind to any structure in the tissues examined.     

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.     

Effects of water stress on enzymes of ammonia assimilation in root nodules of alfalfa (Medicago sativa)

Protein content and activities of the enzymes glutamine synthetase (EC 6.3.1.2), NADH-glutamate synthase (EC 1.4.1.14), NADH-glutamate dehydrogenase (reductive amination (EC 1.4.1.2) and NAD⁺-glutamate dehydrogenase (oxidative deamination) (EC 1.4.1.2) from the plant fraction of root nodules of alfalfa ( Medicago sativa L. cv. Aragon) were determined under water stress. Only NADH-glutamate synthase activity was inhibited during drought. The results indicate that the glutamine synthetase/NADH-glutamate synthase cycle was fully operational in alfalfa nodules of control or even mildly stressed plants when N₂-fixation was not inhibited, but that the coupling between glutamine synthetase and NADH-glutamate synthase was lost as drought progressed. Patterns of glutamine synthetase and NADH-/NAD⁺-gluta-mate dehydrogenase activities reflect changes in ammonia content of nodules and/or availability of carbon substrates, and indicate that nodules maintain sufficient enzyme activity for ammonia assimilation throughout water stress.     

Assimilation of 13NH 4 + by Anthoceros grown with and without symbiotic Nostoc

The pathways of assimilation of ammonium by pure cultures of symbiont-free Anthoceros punctatus L. and the reconstituted Anthoceros-Nostoc symbiotic association were determined from time-course (5–300 s) and inhibitor experiments using ¹³NH ₄ ⁺ . The major product of assimilation after all incubation times was glutamine, whether the tissues were cultured with excess ammonium or no combined nitrogen. The ¹³N in glutamine was predominantly in the amide-nitrogen position. Formation of glutamine and glutamate by Anthoceros-Nostoc was strongly inhibited by either 1mM methionine sulfoximine (MSX) or 1 mM exogenous ammonium. These data are consistent with the assimilation of ¹³NH ₄ ⁺ and formation of glutamate by the glutamine synthetase (EC 6.3.1.2)-glutamate synthase (EC 1.4.7.1) pathway in dinitrogen-grown Anthoceros-Nostoc. However, in symbiont-free Anthoceros, grown with 2.5 mM ammonium, formation of glutamine, but not glutamate, was decreased by either MSX or exogenous ammonium. These results indicate that during short incubation times ammonium is assimilated in nitrogenreplete Anthoceros by the activities of both glutamine synthetase and glutamate dehydrogenase (EC 1.4.1.2). In-vitro activities of glutamine synthetase were similar in nitrogen-replete Anthoceros and Anthoceros-Nostoc, indicating that the differences in the routes of glutamate formation were not based upon regulation of synthesis of the initial enzyme of the glutamine synthetase-glutamate synthase pathway. When symbiont-free Anthoceros was cultured for 2 d in the absence of combined nitrogen, total ¹³NH ₄ ⁺ assimilation, and glutamine and glutamate formation in the presence of inhibitors, were similar to dinitrogen-grown Anthoceros-Nostoc. The routes of immediate (within 2 min) glutamate formation and ammonium assimilation in Anthoceros were apparently determined by the intracellular levels of ammonium; at low levels the glutamine synthetase-glutamate synthase pathway was predominant, while at high levels independent activities of both glutamine synthetase and glutamate dehydrogenase were expressed.     

Pyrimidine nucleotides impair phosphoribosylpyrophosphate (PRPP) synthetase subunit aggregation by sequestering magnesium. A mechanism for the decreased PRPP synthetase activity in hereditary erythrocyte pyrimidine 5′-nucleotidase deficiency

The activity of phosphoribosylpyrophosphate (PRPP) synthetase (ATP:d-ribose-5-phosphate pyrophosphotransferase, EC 2.7.6.1) is decreased in the erythrocyte in hereditary pyrimidine 5′-nucleotidase (P5N) deficiency. Given the increased pyrimidine nucleotide content of the P5N-deficient erythrocyte, we evaluated the effects of prototypic pyrimidine nucleotides on the activity of PRPP synthetase. In normal hemolysate a 1.0 mM combination of cytidine tri-, di-, and monophosphate (CTP/CDP/CMP) inhibited PRPP synthetase activity and changed the ribose 5-phosphate (R5P) saturation curve from a hyperbola to a biphasic shape. Untreated crude hemolysate from P5N-deficient erythrocytes showed a biphasic R5P kinetic curve. Since the activity of PRPP synthetase is dependent on its state of subunit aggregation, we examined PRPP synthetase subunit aggregation using gel permeation chromatography. P5N-deficient erythrocytes had a decreased absolute amount of aggregated PRPP synthetase and almost a total loss of disaggregated PRPP synthetase. Using normal hemolysate, 1 mM CTP/CDP/CMP interfered with the ability of 1.0 mM ATP and 2.0 mM MgCl₂ to promote PRPP synthetase subunit aggregation. Increasing the MgCl₂ to 6.0 mM overcame the inhibitory effect of CTP/CDP/CMP. Thus, the decreased PRPP synthetase activity of the P5N-deficient erythrocyte is due, at least in part, to the ability of the accumulated pyrimidine nucleotides to sequester magnesium and to interfere with the subunit aggregation of PRPP synthetase.     

15N2 Incorporation and metabolism in the lichen Peltigera aphthosa Willd

The Nostoc in the cephalodia of the lichen Peltigera aphthosa Willd. fixed ¹⁵N₂ and the bulk of the nitrogen fixed was continuously transferred from it to its eukaryotic partners (a fungus and a green alga, Coccomyxa sp.). Kinetic studies carried out over the first 30 min, after exposure of isolated cephalodia to ¹⁵N₂, showed that highest initial ¹⁵N₂-labelling was into NH ₄ ⁺ . After 12 min little further increase in the NH ₄ ⁺ label occurred while that in the amide group of glutamine and in glutamate continued to increase. The ¹⁵N-labelling of the amino group of glutamine and of aspartate increased more slowly, followed by an increase in the labelling of alanine. When total incorporation of ¹⁵N-label was calculated, the overall pattern was found to be rather similar except that, throughout the experiment, the total ¹⁵N incorporated into glutamate was about six times greater than that into the amide group of glutamine. Pulse chase experiments, in which ¹⁴N₂ was added to cephalodia previously exposed to ¹⁵N₂, showed that the NH ₄ ⁺ pool rapidly became depleted of ¹⁵N-label, followed by decreases in the labelling of glutamate, the amide group of glutamine and aspartate. The ¹⁵N-labelling of alanine, however, continued to increase for a period. When isolated cephalodia were treated with L-methionine-SR-sulphoximine, an inhibitor of glutamine synthetase (EC 6.3.1.2), and azaserine, an inhibitor of glutamate synthase (EC 2.6.1.53), there was no detectable labelling in glutamine although the ¹⁵N-labelling of glutamate increased unimpaired. On treating the cephalodia with amino-oxyacetate, an inhibitor of aminotransferase activity, the alanine pool decreased. Evidence was obtained that glutamine synthetase and glutamate synthase were located in the Nostoc, and that glutamate dehydrogenase (EC 1.4.1.4) and various amino-transferases were located in the cephalodial fungus. Possible implications of these findings are discussed.Abbreviations MSX L-methionine-SR-sulphoximine - AOA amino-oxyacetate - HEPES N-2-hydroxymethylpiperazine-N-2-ethane sulphonic acid - Tris tris-(hydroxymethyl) methylamine - GS glutamine synthetase - GOGAT glutamate synthase - GDH glutamate dehydrogenase - GPT glutamate-pyruvate aminotransferase - APT aspartate-pyruvate aminotransferase - ADH alanine dehydrogenase - GOT glutamate-oxaloacetate aminotransferase