Synthesis of [N]glutamate from [N]h(4) and [N]glycine by mitochondria isolated from pea and corn shoots

Metabolically competent mitochondria were isolated from pea and corn shoots on Percoll discontinuous density gradients. Rates of synthesis of [¹⁵N]glutamate were measured by gas chromatography-mass spectrometry after the incubation of mitochondria with either 2 millimolar [¹⁵N] H₄⁺ or [¹⁵N]glycine in the presence of 1 millimolar citrate as the respiratory substrate. When [¹⁵N]H₄⁺ was provided, mitochondria isolated from light-grown pea shoots synthesized [¹⁵N]glutamate with a rate of 2.64 nanomoles per hour per milligram mitochondrial protein. Corn mitochondria produced [¹⁵N]glutamate at a rate approximately 11 times greater than the pea mitochondria. Dark treatment during growth for the last 24 hours caused a slight reduction in the rate of synthesis in both species. When [¹⁵N]glycine was used, pea mitochondria synthesized [¹⁵N]glutamate with a rate of 6.32 nanomoles per hour per milligram protein. Rapid disappearance of [¹⁵N]glycine and synthesis of [¹⁵N]serine was observed with a molar ratio of 2 glycine to 0.78 serine. The rate of glutamate synthesis was only 0.2% that of serine, due in part to the dilution of [¹⁵N]H₄⁺ by the [¹⁴N]H₄⁺ pool in the mitochondria. The majority of the [¹⁵N]H₄⁺ released from glycine appears to have been released from or remains unmetabolized in the mitochondria. Corn mitochondria showed no apparent disappearance of [¹⁵N]glycine and little synthesis of [¹⁵N]serine, indicating that our preparation originated primarily from mesophyll cells. Under our conditions of glycine/serine conversion, [¹⁵N]glutatmate was synthesized at a rate of 7% of that of [¹⁵N]serine synthesis by corn mitochondria.     

Simultaneous acquisition of [13C,15N]- and [15N,15N]-separated 4D gradient-enhanced NOESY spectra in proteins

Summary The simultaneous acquisition of a 4D gradient-enhanced and sensitivity-enhanced [¹³C,¹⁵N]/[¹⁵N,¹⁵N]-separated NOESY is presented for the 74-residue [¹³C,¹⁵N]-labeled N-terminal SH3 domain of mGrb2 complexed with a peptide gragment from mSOS-2 in 90% H₂O. The method readily accommodates different ¹³C and ¹⁵N spectral widths, but requires that the same number of increments be collected for both ¹³C and ¹⁵N in the simultaneous dimension (F₂). For purposes of display and analysis, the two 4D spectra can be deconvolved during the processing stage by the appropriate linear combination of separately stored FIDs. Compared to collecting each of these two 4D data sets separately, the presented method is a factor (2)^1/2 more efficient in sensitivity per unit acquisition time. The interleaved nature of this method may also lead to improved peak registration between the two 4D spectra.     

Nitrogen Dynamics in Stream Wood Samples Incubated with [14C]Lignocellulose and Potassium [15N]Nitrate

Surface wood samples obtained from a Douglas fir log (Pseudotsuga menziesii) in a Pacific Northwest stream were incubated in vitro with [¹⁴C]lignocellulose in a defined mineral salts medium supplemented with 10 mg of N liter⁻¹ of ¹⁵N-labeled NO₃⁻ (50 atom% ¹⁵N). Evolution of ¹⁴CO₂, distribution and isotopic dilution of ¹⁵N, filtrate N concentrations, and the rates of denitrification, N₂ fixation, and respiration were measured at 6, 12, and 18 days of incubation. The organic N content of the lignocellulose-wood sample mixture had increased from 132 μg of N to a maximum of 231 μg of N per treatment after 6 days of incubation. Rates of [¹⁴C]lignocellulose decomposition were greatest during the first 6 days and then began to decline over the remaining 12 days. Total CO₂ evolution was also highest at day 6 and declined steadily over the remaining duration of the incubation. Filtrate NH₄⁺-N increased from background levels to a final value of 57 μg of N per treatment. Filtrate NO₃⁻ N completely disappeared by day 6, and organic N showed a slight decline between days 12 and 18. The majority of the ¹⁵N that could be recovered appeared in the particulate organic fraction by day 6 (41 μg of N), and the filtrate NH₄⁺ N fraction contained 11 μg of ¹⁵N by day 18. The ¹⁵N enrichment values of the filtrate NH₄⁺ and the inorganic N associated with the particulate fraction had increased to approximately 20 atom% ¹⁵N by 18 days of incubation, whereas the particulate organic fraction reached its highest enrichment by day 6. Measurements of N₂ fixation and denitrification indicated an insignificant gain or loss of N from the experimental system by these processes. The data show that woody debris in stream ecosystems might function as a rapid and efficient sink for exogenous N, resulting in stimulation of wood decomposition and subsequent activation of other N cycling processes.     

Metabolism of [15N]Alanine in the Ectomycorrhizal Fungus Paxillus involutus

Chalot, M., Finlay, R. D., Ek, H., and Söderström, B. 1995. Metabolism of [¹⁵N]alanine in the ectomycorrhizal fungus Paxillus involutus. Experimental Mycology 19, 297-304. Alanine metabolism in the ectomycorrhizal fungus Paxillus involutus was investigated using [¹⁵N]alanine. Short-term exposure of mycelial discs to [¹⁵N]alanine showed that the greatest flow of ¹⁵N was to glutamate and to aspartate. Levels of enrichment were as high as 15-20% for glutamate and 13-18% for aspartate, whereas that of alanine reached 30%. Label was also detected in the amino-N of glutamine and in serine and glycine, although at lower levels. Preincubation of mycelia with aminooxyacetate, an inhibitor of transamination reactions. resulted in complete inhibition of the flow of the label to glutamate, aspartate, and amino-N of glutamine, whereas [¹⁵N]alanine rapidly accumulated. This evidence indicates the direct involvement of alanine aminotransferase for translocation of ¹⁵N from alanine to glutamate. Alanine may be a convenient reservoir of both nitrogen and carbon.     

Gas chromatography-mass spectrometry determination of [15N]ammonia enrichment in blood and urine

A rapid gas chromatography-mass spectrometry method for [¹⁵N]ammonia analysis is deseribed which is based on the formation of [¹⁵N]glutamic acid from ammonia and analysis of isotopic abundance in the N-trifluoroacetyl-n-butylester glutamate derivative. Mean recovery of [¹⁵N]ammonia added to either plasma or urine was greater than 99% with a relative standard deviation of less than 10%. The method can be applied to the determination of extremely low levels of ammonia through an isotope dilution technique. The [¹⁵N]ammonia abundance of blood and urine was determined in an adult following on oral dose (500 mg) of ¹⁵NH₄Cl. A peak isotopic abundance of 13 atoms% excess was reached by 30 min. Urinary excretion of [¹⁵N]ammonia during the first 4 h after administration of the isotope amounted to 4.1% of the isotope administered.     

Specifically alkylated DNA fragments. Synthesis and physical characterization of d[CGC(O6Me)GCG] and d[CGT(O6Me)GCG]

Two hexamer DNA fragments containing a carcinogenic modified base, O⁶-methyl guanine, have been synthesized by a solid-phase phosphotriester method, in which the unmodified guanine residues present were O⁶ protected with the 4-nitrophenylethyl group. These two alkylated oligonucleotides were found to have similar T_m's about 40° lower than the unmodified parent compund, d(CG)₃. Moreover, the presence of the (O⁶Me)G appears to inhibit the B→Z transition, as determined by CD spectroscopy.     

Resonance assignment and structural analysis of acid denatured E. coli [U-15N]-glutaredoxin 3

Virtually complete sequence specific ¹H and ¹⁵N resonance assignments are presented for acid denatured reduced E. coli glutaredoxin 3. The sequential resonance assignments of the backbone rely on the combined use of 3D F₁-decoupled ROESY-¹⁵N-HSQC and 3D ¹⁵N-HSQC-(TOCSY-NOESY)-¹⁵N-HSQC using a single uniformly ¹⁵N labelled protein sample. The sidechain resonances were assigned from a 3D TOCSY-¹⁵N-HSQC and a homonouclear TOCSY spectrum. The presented assignment strategy works in the absence of chemical exchange peaks with signals from the native conformation and without ¹³C/¹⁵N double labelling. Chemical shifts, ³J(H, NH) coupling constants and NOEs indicate extensive conformational averaging of both backbone and side chains in agreement with a random coil conformation. The only secondary structure element persisting at pH 3.5 appears to be a short helical segment comprising residues 37 to 40.Abbreviations HSQC heteronuclear single quantum coherence - NMR nuclear magnetic resonance - NOE nuclear Overhauser effect - NOESY two-dimensional NOE spectroscopy - ROE nuclear Overhauser effect in the rotating frame - ROESY two-dimensional ROE spectroscopy - TOCSY total correlation spectroscopy - TPPI time proportional phase incrementation Correspondence to: G. Otting     

Regiospecipic Synthesis of a New Cross-Linked Dinucleoside : 1-(N6-Deoxyadenyl)-2-(o4-Thymidyl)-Ethane

Abstract

The bridged dinucleoside 1-(N⁶-deoxyadenyl) 2-(O⁴-thymidyl)-ethane was prepared from the nucleophilic substitution of a O⁴-triazo-lyl thymidine by a N⁶-(2-hydroxyethyl) deoxyadenosine derivative via the corresponding 6-halogeno hypoxanthine in ribose and deoxyribose series.     

Improved sensitivity and coherence selection for [15N,1H]-TROSY elements in triple resonance experiments

In experiments with proteins of molecular weights around 100 kDa the implementation of [¹⁵N,¹H]-TROSY-elements in [¹⁵N]-constant-time triple resonance experiments yields sensitivity enhancements of one to two orders of magnitude. An additional gain of 10 to 20% may be obtained with the use of sensitivity enhancement elements. This paper describes a novel sensitivity enhancement scheme which is based on concatenation of the ¹³ C ¹⁵N magnetization transfer with the ST2-PT element, and which enables proper TROSY selection of the ¹⁵N multiplet components.     

INCORPORATION OF 8-HISTAMINYL-DEOXYADENOSINE [8-(2-(4-IMIDAZOLYL)ETHYLAMINO)-2′-DEOXYRIBOADENOSINE] INTO OLIGODEOXYRIBONUCLEOTIDES BY SOLID PHASE PHOSPHORAMIDITE COUPLING

ABSTRACT

The 3′phosphoramidite of 8-histaminyl deoxyadenosine has been prepared and successfully incorporated into a short oligodeoxyribonucleotide. The synthetic methodology leading to this preparation is given and the implications for developing new DNAzymes as well as probing unusual nucleic acid structures are discussed.     

Towards high-resolution solid-state NMR on large uniformly 15N- and [13C,15N]-labeled membrane proteins in oriented lipid bilayers

Based on exact numerical simulations, taking into account isotropic and conformation-dependent anisotropic nuclear spin interactions, we systematically analyse the prospects for high-resolution solid-state NMR on large isotope-labeled membrane proteins macroscopically oriented in phospholipid bilayers. Using the known X-ray structures of rhodopsin and porin as models for large membrane proteins with typical -helical and -barrel structural motifs, the analysis considers all possible one- to six-dimensional spectra comprised of frequency dimensions with evolution under any combination of amide ¹H, amide ¹⁵N, and carbonyl ¹³C chemical shifts as well as ¹H-¹⁵N dipole-dipole couplings. Under consideration of typical nuclear spin interaction and experimental line-shape parameters, the analysis provides new insight into the resolution capability and orientation-dependent transfer efficiency of existing experiments as well as guidelines as to improved experimental approaches for the study of large uniformly ¹⁵N- and [¹³C,¹⁵N]-labeled membrane proteins. On basis of these results and numerical optimizations of coherence-transfer efficiencies, we propose several new high-resolution experiments for sequential protein backbone assignment and structure determination.     

1H and 15N NMR studies of protonation and hydrogen-bonding in the binding of trimethoprim to dihydrofolate reductase

The binding of trimethoprim and [1,3,2-amino-¹⁵N₃]-trimethoprim to Lactobacillus casei dihydrofolate reductase has been studied by ¹⁵N and ¹H NMR spectroscopy. ¹⁵N NMR spectra of the bound drug were obtained by using polarisation transfer pulse sequences. The ¹⁵N chemical shifts and ¹H-¹⁵N spin-coupling constants show unambiguously that the drug is protonated on N1 when bound to the enzyme.The N1-proton resonance in the complex has been assigned using the ¹⁵N-enriched molecule. The temperature-dependence of the linewidth of this resonance has been used to estimate the rate of exchange of this proton with the solvent: 160±10s^-1 at 313 K, with an activation energy of 75 (±9) kJ·mole^-1. This is considerably faster than the dissociation rate of the drug from this complex, demonstrating that there are local fluctuations in the structure of the complex.     

[15N] leucine as a source of [15N] glutamate in organotypic cerebellar explants

Approximately 26.0% of the [¹⁵N] glutamate and [alpha ¹⁵N] glutamine formed in organotypic cerebellar explants was derived from [¹⁵N] leucine. Approximately 14.0% of the ¹⁵NH₃ and [amide ¹⁵N] glutamine synthesized came from leucine nitrogen. Another 4.0% of the alpha nitrogen of both glutamate and glutamine was derived from [¹⁵N] valine. These results suggest that branched-chain amino acids, particularly leucine, may be important for the synthesis of glutamic acid by the brain.     

Gas Chromatography-Mass Spectrometry of N- Heptafluorobutyryl Isobutyl Esters of Amino Acids in the Analysis of the Kinetics of [N]H(4) Assimilation in Lemna minor L

Rapid, sensitive, and selective methods for the determination of the ¹⁵N abundance of amino acids in isotopic tracer experiments with plant tissues are described and discussed. Methodology has been directly tested in an analysis of the kinetics of [¹⁵N]H₄⁺ assimilation in Lemna minor L. The techniques utilize gas chromatography-mass spectrometry selected ion monitoring of major fragments containing the N moiety of N-heptafluorobutyryl isobutyl esters of amino acids. The ratio of selected ion pairs at the characteristic retention time of each amino acid derivative can be used to calculate ¹⁵N abundance with an accuracy of ±1 atom% excess ¹⁵N using samples containing as little as 30 picomoles of individual amino acids. Up to 11 individual amino acid derivatives can be selectively monitored in a single chromatogram of 30 minutes. It is suggested that these techniques will be useful in situations where the small quantities of N available for analysis have hitherto hindered the use of ¹⁵N-labeled precursors.     

Assimilation of [N]Nitrate and [N]Nitrite in Leaves of Five Plant Species under Light and Dark Conditions

Light dependency of nitrate and nitrite assimilation to reduced-N in leaves remains a controversial issue in the literature. With the objective of resolving this controversy, the light requirement for nitrate and nitrite assimilation was investigated in several plant species. Dark and light assimilation of [¹⁵N]nitrate and [¹⁵N]nitrite to ammonium and amino-N was determined with leaves of wheat, corn, soybean, sunflower, and tobacco. In dark aerobic conditions, assimilation of [¹⁵N]nitrate as a percentage of the light rate was 16 to 34% for wheat, 9 to 16% for tobacco, 26% for corn, 35 to 76% for soybean, and 55 to 63% for sunflower. In dark aerobic conditions, assimilation of [¹⁵N]nitrite as a percentage of the light rate was 11% for wheat, 7% for tobacco, 13% for corn, 28 to 36% for soybeans, and 12% for sunflower. It is concluded that variation among plant species in the light requirement for nitrate and nitrite assimilation explains some of the contradictory results in the literature, but additional explanations must be sought to fully resolve the controversy.

In dark anaerobic conditions, the assimilation of [¹⁵N]nitrate to ammonium and amino-N in leaves of wheat, corn, and soybean was 43 to 58% of the dark aerobic rate while dark anaerobic assimilation of [¹⁵N]nitrite for the same species was 31 to 41% of the dark aerobic rate. In contrast, accumulation of nitrite in leaves of the same species in the dark was 2.5-to 20-fold higher under anaerobic than aerobic conditions. Therefore, dark assimilation of nitrite cannot alone account for the absence of nitrite accumulation in the in vivo nitrate reductase assay under aerobic conditions. Oxygen apparently inhibits nitrate reduction in the dark even in leaves of plant species that exhibit a relatively high dark rate of [¹⁵N]nitrite assimilation.

     

Improved 3D triple resonance experiments, HNN and HN(C)N, for HN and 15N sequential correlations in (13C, 15N) labeled proteins: application to unfolded proteins

Two triple resonance experiments, HNN and HN(C)N, are presented which correlate H^N and ¹⁵N resonances sequentially along the polypeptide chain of a doubly (¹³C, ¹⁵N) labeled protein. These incorporate several improvements over the previously published sequences for a similar purpose and have several novel features. The spectral characteristics enable direct identification of certain triplets of residues, which provide many starting points for the sequential assignment procedure. The experiments are sensitive and their utility has been demonstrated with a 22 kDa protein under unfolding conditions where most of the standard triple resonance experiments such as HNCA, CBCANH etc. have limited success because of poor amide, C and C chemical shift dispersions.     

Relative and accurate measurement of protein abundance using 15N stable isotope labeling in Arabidopsis (SILIA)

In the quantitative proteomic studies, numerous in vitro and in vivo peptide labeling strategies have been successfully applied to measure differentially regulated protein and peptide abundance. These approaches have been proven to be versatile and repeatable in biological discoveries. ¹⁵N metabolic labeling is one of these widely adopted and economical methods. However, due to the differential incorporation rates of ¹⁵N or ¹⁴N, the labeling results produce imperfectly matched isotopic envelopes between the heavy and light nitrogen-labeled peptides. In the present study, we have modified the solid Arabidopsis growth medium to standardize the ¹⁵N supply, which led to a uniform incorporation of ¹⁵N into the whole plant protein complement. The incorporation rate (97.43 ± 0.11%) of ¹⁵N into ¹⁵N-coded peptides was determined by correlating the intensities of peptide ions with the labeling efficiencies according to Gaussian distribution. The resulting actual incorporation rate (97.44%) and natural abundance of ¹⁵N/¹⁴N-coded peptides are used to re-calculate the intensities of isotopic envelopes of differentially labeled peptides, respectively. A modified ¹⁵N/¹⁴N stable isotope labeling strategy, SILIA, is assessed and the results demonstrate that this approach is able to differentiate the fold change in protein abundance down to 10%. The machine dynamic range limitation and purification step will make the precursor ion ratio deriving from the actual ratio fold change. It is suggested that the differentially mixed ¹⁵N-coded and ¹⁴N-coded plant protein samples that are used to establish the protein abundance standard curve should be prepared following a similar protein isolation protocol used to isolate the proteins to be quantitated.     

15N resonance assignments of oxidized and reducedChromatium vinosum high-potential iron protein

The¹⁵N resonances in reduced and oxidizedChromatium vinosum high-potential iron protein have been assigned by use of¹H-¹H COSY spectra and¹H-¹⁵N HMQC, HMQC-COSY, and HMQC-NOESY spectra. Unambiguous assignment of 70 of 85 backbone¹⁵N resonances in the reduced protein and 62 of 85 resonances in the oxidized protein are made, as are 12 of 21 side-chain¹⁵N resonances.     

Nitrogen fixation in legumes and actinorhizal plants in natural ecosystems: values obtained using 15N natural abundance

Background: Nitrogen fixation has been quantified for a range of crop legumes and actinorhizal plants under different agricultural/agroforestry conditions, but much less is known of legume and actinorhizal plant N₂ fixation in natural ecosystems.

Aims: To assess the proportion of total plant N derived from the atmosphere via the process of N₂ fixation (%Ndfa) by actinorhizal and legume plants in natural ecosystems and their N input into these ecosystems as indicated by their ¹⁵N natural abundance.

Methods: A comprehensive collation of published values of %Ndfa for legumes and actinorhizal plants in natural ecosystems and their N input into these ecosystems as estimated by their ¹⁵N natural abundance was carried out by searching the ISI Web of Science database using relevant key words.

Results: The %Ndfa was consistently large for actinorhizal plants but very variable for legumes in natural ecosystems, and the average value for %Ndfa was substantially greater for actinorhizal plants. High soil N, in particular, but also low soil P and water content were correlated with low legume N₂ fixation. N input into ecosystems from N₂ fixation was very variable for actinorhizal and legume plants and greatly dependent on their biomass within the system.

Conclusions: Measurement of ¹⁵N natural abundance has given greater understanding of where legume and actinorhizal plant N₂ fixation is important in natural ecosystems. Across studies, the average value for %Ndfa was substantially greater for actinorhizal plants than for legumes, and the relative abilities of the two groups of plants to utilise mineral N requires further study.     

1H(C) and 1H(N) total NOE correlations in a single 3D NMR experiment. 15N and 13C time-sharing in t1 and t2 dimensions for simultaneous data acquisition

Simultaneous data acquisition in time-sharing (TS) multi-dimensional NMR experiments has been shown an effective means to reduce experimental time, and thus to accelerate structure determination of proteins. This has been accomplished by spin evolution time-sharing of the X and Y heteronuclei, such as ¹⁵N and ¹³C, in one of the time dimensions. In this work, we report a new 3D TS experiment, which allows simultaneous ¹³C and ¹⁵N spin labeling coherence in both t ₁ and t ₂ dimensions to give four NOESY spectra in a single 3D experiment. These spectra represent total NOE correlations between ¹H_N and ¹H_C resonances. This strategy of double time-sharing (2TS) results in an overall four-fold reduction in experimental time compared with its conventional counterpart. This 3D 2TS CN-CN-H HSQC-NOESY-HSQC pulse sequence also demonstrates improvements in water suppression, ¹⁵N spectral resolution and sensitivity, which were developed based on 2D TS CN-H HSQC and 3D TS H-CN-H NOESY-HSQC experiments. Combining the 3D TS and the 3D 2TS NOESY experiments, NOE assignment ambiguities and errors are considerably reduced. These results will be useful for rapid protein structure determination to complement the effort of discerning the functions of diverse genomic proteins.