Source and Magnitude of Ammonium Generation in Maize Roots1

Studies with 15N indicate that appreciable generation of NH4+ from endogenous sources accompanies the uptake and assimilation of exogenous NH4+ by roots. To identify the source of NH4+ generation, maize (Zea mays L.) seedlings were grown on 14NH4+ and then exposed for 3 d to highly labeled 15NH4+. More of the entering 15NH4+ was incorporated into the protein-N fraction of roots in darkness (approximately 25%) than in the light (approximately 14%). Although the 14NH4+ content of roots declined rapidly to less than 1 μmol per plant, efflux of 14NH4+ continued throughout the 3-d period at an average daily rate of 14 μmol per plant. As a consequence, cumulative 14NH4+ efflux during the 3-d period accounted for 25% of the total 14N initially present in the root. Although soluble organic 14N in roots declined during the 3-d period, insoluble 14N remained relatively constant. In shoots both soluble organic 14N and 14NH4+ declined, but a comparable increase in insoluble 14N was noted. Thus, total 14N in shoots remained constant, reflecting little or no net redistribution of 14N between shoots and roots. Collectively, these observations reveal that catabolism of soluble organic N, not protein N, is the primary source of endogenous NH4+ generation in maize roots.

Preferential expression of an ammonium transporter and of two putative nitrate transporters in root hairs of tomato.

Root hairs as specialized epidermal cells represent part of the outermost interface between a plant and its soil environment. They make up to 70% of the root surface and, therefore, are likely to contribute significantly to nutrient uptake. To study uptake systems for mineral nitrogen, three genes homologous to Arabidopsis nitrate and ammonium transporters (AtNrt1 and AtAmt1) were isolated from a root hair-specific tomato cDNA library. Accumulation of LeNrt1-1, LeNrt1-2, and LeAmt1 transcripts was root-specific, with no detectable transcripts in stems or leaves. Expression was root cell type-specific and regulated by nitrogen availability. LeNrt1-2 mRNA accumulation was restricted to root hairs that had been exposed to nitrate. In contrast, LeNrt1-1 transcripts were detected in root hairs as well as other root tissues under all nitrogen treatments applied. Analogous to LeNrt1-1, the gene LeAmt1 was expressed under all nitrogen conditions tested, and root hair-specific mRNA accumulation was highest following exposure to ammonium. Expression of LeAMT1 in an ammonium uptake-deficient yeast strain restored growth on low ammonium medium, confirming its involvement in ammonium transport. Root hair specificity and characteristics of substrate regulation suggest an important role of the three genes in uptake of mineral nitrogen.

Probing the oligomeric structure of an enzyme by electrospray ionization time-of-flight mass spectrometry.

Electrospray ionization time-of-flight (ESI-TOF) mass spectrometry was used to study the quaternary structure of 4-oxalocrotonate tautomerase (EC 5.3.2; 4OT), and four analogues prepared by total chemical synthesis. Wild-type 4OT is a hexamer of 62 amino acid subunits and contains no cysteine residues. The analogues were: (desPro1)4OT, a truncated construct in which Pro1 was deleted; (Cpc1)4OT in which Pro1 was replaced with cyclopentane carboxylate; a derivative [Met(O)45]4OT in which Met45 was oxidized to the sulfoxide; and an analogue (Nle45)4OT in which Met45 was replaced with norleucine. ESI of (Nle45)4OT, (Cpc1)4OT, and 4OT from solution conditions under which the native enzyme was fully active (5 mM ammonium bicarbonate buffer, pH 7.5) gave the intact hexamer as the major species detected by TOF mass spectrometry. In contrast, analysis of [Met(O)45]4OT and (desPro1)4OT under similar conditions yielded predominantly monomer ions. The ESI-TOF measurements were consistent with structural data obtained from circular dichroism spectroscopy. In the context of kinetic data collected for 4OT and these analogues, ESI-TOF mass spectrometry also provided important evidence for the structural and mechanistic significance of the catalytically important Pro1 residue in 4OT.

Excitation transfer spectroscopy with two metastable 138Ba+ ions in same trap.

In the absence of lasers approaching trapped ion clock transitions in sharpness we propose to replace the 12.49 m laser field exciting the D3/2-D5/2 transition of the single Ba+ ion A in D3/2 with the near-field of a close by identical ion B in the excited D5/2 state. We tune the frequency of the near-field by the differential Stark shift generated when the center of mass of the tuned ions is slightly moved out of the trap center by a small bias voltage. We demonstrate that the resultant resonant energy exchange can be made considerably faster than the natural lifetime of either metastable level and show how it might be detected.

Conformations and folding of lysozyme ions in vacuo.

Proton transfer reactivity of isolated charge states of the protein hen egg-white lysozyme shows that multiple distinct conformations of this protein are stable in the gas phase. The reactivities of the 9+ and 10+ charge state ions, formed by electrospray ionization of "native" (disulfide-intact) and "denatured" (disulfide-reduced) solutions, are consistent with values calculated for ions in their crystal structure and fully denatured conformations, respectively. Charge states below 8+ of both forms, formed by proton stripping, have similar or indistinguishable reactivities, indicating that the disulfide-reduced ions fold in the gas phase to a more compact conformation.

Mammalian phospholipase D: activation by ammonium sulfate and nucleotides.

Phospholipase D (PLD) associated with the rat kidney membrane was activated by guanine 5'-[gamma-thio]triphosphate and a cytosol fraction that contained ADP-ribosylation factor. When assayed by measuring the phosphatidyl transfer reaction to ethanol with exogenously added radioactive phosphatidylcholine as substrate, the PLD required a high concentration (1.6 M) of ammonium sulfate to exhibit high enzymatic activity. Other salts examined were far less effective or practically inactive, and this dramatic action of ammonium sulfate is not simply due to such high ionic strength. Addition of ATP but not of nonhydrolyzable ATP analogue adenosine 5'-[beta, gamma-imido]diphosphate further enhanced the PLD activation approximately equal to 2- to 3-fold. This enhancement by ATP needed cytosol, implying a role of protein phosphorylation. A survey of PLD activity in rat tissues revealed that, unlike in previous observations reported thus far, PLD was most abundant in membrane fractions of kidney, spleen, and liver in this order, and the enzymatic activity in brain and lung was low.

Crystallographic evidence for the action of potassium, thallium, and lithium ions on fructose-1,6-bisphosphatase.

Fructose-1,6-bisphosphatase (Fru-1,6-Pase; D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) requires two divalent metal ions to hydrolyze alpha-D-fructose 1,6-bisphosphate. Although not required for catalysis, monovalent cations modify the enzyme activity; K+ and Tl+ ions are activators, whereas Li+ ions are inhibitors. Their mechanisms of action are still unknown. We report here crystallographic structures of pig kidney Fru-1,6-Pase complexed with K+, Tl+, or both Tl+ and Li+. In the T form Fru-1,6-Pase complexed with the substrate analogue 2,5-anhydro-D-glucitol 1,6-bisphosphate (AhG-1,6-P2) and Tl+ or K+ ions, three Tl+ or K+ binding sites are found. Site 1 is defined by Glu-97, Asp-118, Asp-121, Glu-280, and a 1-phosphate oxygen of AhG-1,6-P2; site 2 is defined by Glu-97, Glu-98, Asp-118, and Leu-120. Finally, site 3 is defined by Arg-276, Glu-280, and the 1-phosphate group of AhG-1,6-P2. The Tl+ or K+ ions at sites 1 and 2 are very close to the positions previously identified for the divalent metal ions. Site 3 is specific to K+ or Tl+. In the divalent metal ion complexes, site 3 is occupied by the guanidinium group of Arg-276. These observations suggest that Tl+ or K+ ions can substitute for Arg-276 in the active site and polarize the 1-phosphate group, thus facilitating nucleophilic attack on the phosphorus center. In the T form complexed with both Tl+ and Li+ ions, Li+ replaces Tl+ at metal site 1. Inhibition by lithium very likely occurs as it binds to this site, thus retarding turnover or phosphate release. The present study provides a structural basis for a similar mechanism of inhibition for inositol monophosphatase, one of the potential targets of lithium ions in the treatment of manic depression.

Coordinate regulation of Bacillus subtilis peroxide stress genes by hydrogen peroxide and metal ions.

The Bacillus subtilis mrgA gene encodes an abundant DNA-binding protein that protects cells against the lethal effects of H2O2. Transcription of mrgA is induced by H2O2 or by entry into stationary phase when manganese and iron levels are low. We have selected for strains derepressed for transcription of mrgA in the presence of Mn(II). The resulting cis-acting mutants define an operator site just upstream of the mrgA promoter. Similar sequences flank the promoters for the catalase gene, katA, and the heme biosynthesis operon, hemAXCDBL. Like mrgA, transcription of the katA and hem genes is repressed by Mn(II), which thereby potentiates the killing action of H2O2. We identified two classes of trans-acting mutants derepressed for mrgA transcription in the presence of Mn(II): some exhibit a coordinate derepression of MrgA, catalase, heme biosynthesis, and alkyl hydroperoxide reductase and are H2O2 resistant, while others have reduced catalase activity and are H2O2 sensitive. These data indicate that the peroxide stress response of B. subtilis is regulated by a repressor that senses both metal ion levels and H2O2.

Sticky ions in biological systems.

Aqueous gel sieving chromatography on Sephadex G-10 of the Group IA cations (Li+, Na+, K+, Rb+, Cs+) plus NH4+ as the Cl- salts, in combination with previous results for the halide anions (F-, Cl-, Br-, I-) as the Na+ salts [Washabaugh, M.W. & Collins, K.D. (1986) J. Biol. Chem. 261, 12477-12485], leads to the following conclusions. (i) The small monovalent ions (Li+, Na+, F-) flow through the gel with water molecules attached, whereas the large monovalent ions (K+, Rb+, Cs+, Cl-, Br-, I-) adsorb to the nonpolar surface of the gel, a process requiring partial dehydration of the ion and implying that these ions bind the immediately adjacent water molecules weakly. (ii) The transition from strong to weak hydration occurs at a radius of about 1.78 A for the monovalent anions, compared with a radius of about 1.06 A for the monovalent cations (using ionic radii), indicating that the anions are more strongly hydrated than the cations for a given charge density. (iii) The anions show larger deviations from ideal behavior (an elution position corresponding to the anhydrous molecular weight) than do the cations and dominate the chromatographic behavior of the neutral salts. These results are interpreted to mean that weakly hydrated ions (chaotropes) are "pushed" onto weakly hydrated surfaces by strong water-water interactions and that the transition from strong ionic hydration to weak ionic hydration occurs where the strength of ion-water interactions approximately equals the strength of water-water interactions in bulk solution.

Role of sodium and potassium ions in regulation of glucose metabolism in cultured astroglia.

Effects of increasing extracellular K+ or intracellular Na+ concentrations on glucose metabolism in cultures of rat astroglia and neurons were examined. Cells were incubated in bicarbonate buffer, pH 7.2, containing 2 mM glucose, tracer amounts of [14C]deoxyglucose ([14C]dGlc), and 5.4, 28, or 56 mM KCl for 10, 15, or 30 min, and then for 5 min in [14C]dGlc-free buffer to allow efflux of unmetabolized [14C]dGlc. Cells were then digested and assayed for labeled products, which were shown to consist of 96-98% [14C]deoxyglucose 6-phosphate. Increased K+ concentrations significantly raised [14C]deoxyglucose 6-phosphate accumulation in both neuronal and mixed neuronal-astroglial cultures at 15 and 30 min but did not raise it in astroglial cultures. Veratridine (75 microM), which opens voltage-dependent Na+ channels, significantly raised rates of [14C]dGlc phosphorylation in astroglial cultures (+20%), and these elevations were blocked by either 1 mM ouabain, a specific inhibitor of Na+,K(+)-ATPase (EC 3.6.1.37), or 10 microM tetrodotoxin, which blocks Na+ channels. The carboxylic sodium ionophore, monensin (10 microM), more than doubled [14C]dGlc phosphorylation; this effect was only partially blocked by ouabain and unaffected by tetrodotoxin. L-Glutamate (500 microM) also stimulated [14C]dGlc phosphorylation in astroglia--not through N-methyl-D-aspartate or non-N-methyl-D-aspartate receptor mechanisms but via a Na(+)-dependent glutamate-uptake system. These results indicate that increased uptake of Na+ can stimulate energy metabolism in astroglial cells.

Complexos de vanádio como miméticos de peroxidases e catalisadores de oxidação: síntese e caracterização espectroscópica

Quatro novos complexos mononucleares e um dinuclear de vanádio(IV) contendo ligantes do tipo imínico e carboxilato foram sintetizados e caracterizados através de diferentes técnicas espectroscópicas, UVNis, FT/IR e EPR, além de análise elementar, medidas de condutividade molar e, para alguns deles, análise termogravimétrica. Alguns deles foram obtidos como espécies neutras e outros, contendo grupos carboxilatos, foram isolados como espécies aniônicas, com contra-íons Na+ ou NH4+. Os complexos clássicos da literatura, largamente estudados e caracterizados, [VIVO(acac)2] e [VIvO(salen)], também foram sintetizados e caracterizados, a fim de comparar suas propriedades com aquelas dos novos complexos de vanádio(IV) sintetizados. Através das técnicas espectroscópicas, as principais bandas de transição e os principais grupos funcionais existentes nos complexos puderam ser verificados, bem como a simetria da estrutura geométrica e a confirmação do estado de oxidação do metal nos complexos. Além disso, através de medidas de condutividade molar e análise térmica foram confirmadas as razões estequiométricas ligante: metal em cada complexo, verificando-se, por exemplo, a natureza dimérica proposta para o complexo [VIVO(dbhab)] 2.

Nitrogen regulation of protein–protein interactions and transcript levels of GlnK PII regulator and AmtB ammonium transporter homologs in Archaea

Gene homologs of GlnK PII regulators and AmtB-type ammonium transporters are often paired on prokaryotic genomes, suggesting these proteins share an ancient functional relationship. Here, we demonstrate for the first time in Archaea that GlnK associates with AmtB in membrane fractions after ammonium shock, thus, providing a further insight into GlnK-AmtB as an ancient nitrogen sensor pair. For this work, Haloferax mediterranei was advanced for study through the generation of a pyrE2-based counterselection system that was used for targeted gene deletion and expression of Flag-tagged proteins from their native promoters. AmtB1-Flag was detected in membrane fractions of cells grown on nitrate and was found to coimmunoprecipitate with GlnK after ammonium shock. Thus, in analogy to bacteria, the archaeal GlnK PII may block the AmtB1 ammonium transporter under nitrogen-rich conditions. In addition to this regulated protein–protein interaction, the archaeal amtB-glnK gene pairs were found to be highly regulated by nitrogen availability with transcript levels high under conditions of nitrogen limitation and low during nitrogen excess. While transcript levels of glnK-amtB are similarly regulated by nitrogen availability in bacteria, transcriptional regulators of the bacterial glnK promoter including activation by the two-component signal transduction proteins NtrC (GlnG, NRI) and NtrB (GlnL, NRII) and sigma factor σN (σ54) are not conserved in archaea suggesting a novel mechanism of transcriptional control.

Enantioselective alkylation of β-keto esters promoted by dimeric Cinchona-derived ammonium salts as recoverable organocatalysts

Dimeric anthracenyldimethyl-derived Cinchona ammonium salts are used as chiral organocatalysts in 5 mol% for the phase-transfer enantioselective alkylation reaction of 2-alkoxycarbonyl-1-indanones with activated bromides. The corresponding adducts bearing a new all-carbon quaternary center are obtained usually in high yield and with moderate and opposite enantioselectivity (up to 55%) when using ammonium salts derived from quinidine and its pseudoenantiomer quinine as organocatalysts. These catalysts can be almost quantitatively recovered by precipitation in ether and reused.

The contribution of sulfate ions and protons to the specific capacitance of microporous carbon monoliths

The monoliths studied in this work show large specific surface areas (up to 1600 m2 g-1), high densities (up to 1.17 g cm-3) and high electrical conductivities (up to 9.5 S cm-1). They are microporous carbons with pore sizes up to 1.3 nm but most of them below 0.75 nm. They also show oxygen functionalities. The electrochemical behavior of the monoliths is studied in three-electrode cells with aqueous H2SO4 solution as electrolyte. This work deals with the contribution of the sulfate ions and protons to the specific capacitance of carbon monoliths having different surface areas and different contents of oxygen groups. Protons contribute with a pseudocapacitance (up to 152 F g-1) in addition to the double layer capacitance. Sulfate ions contribute with a double layer capacitance only. At the double layer, the capacitance of the sulfate ions (up to 291 F g-1) is slightly higher than that of protons (up to 251 F g-1); both capacitances increase as the surface area increases. The preference of protons to be electroadsorbed at the double layer and the broader voltage window of these ions account for their higher contribution (70 %) to the double layer capacitance.

Ferredoxin-dependent glutamate synthase: involvement in ammonium assimilation in Haloferax mediterranei

Glutamate synthase (GOGAT) is one of the two important enzymes involved in the ammonium assimilation pathway glutamine synthetase (GS)/GOGAT, which enables Hfx. mediterranei to thrive in media with low ammonium concentration or containing just nitrate as single nitrogen source. The gene coding for this enzyme, gltS, has been sequenced, analysed and compared with other GOGATs from different organisms from the three domains of life. According to its amino acid sequence, Hfx. mediterranei GOGAT displays high homology with those from other archaeal halophilic organisms and with the bacterial alpha-like subunit. Hfx. mediterranei GOGAT and GS expression was induced under conditions of ammonium restriction. The GOGAT protein was found to be a monomer with a molecular mass of 163.78 kDa, which is consistent with that estimated by gel filtration, 198 ± 30 kDa. The enzyme is highly ferredoxin dependent: activity was only observed with one of the two different 2Fe–2S ferredoxins chromatographically isolated from Hfx. mediterranei. The enzyme also displayed typical halophilic behaviour, being fully stable, and producing maximal activity, at salt concentrations from 3 to 4 M NaCl, pH 7.5 and a temperature of 50 °C.