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1.
A new method which utilizes 13C- 13C coupling for structural and biosynthetic studies on acetate-derived metabolites is described. The 13C-NMR spectra of dihydrolatumcidins separately labeled with 13CH 313C0 2Na and with a 1: 1 mixture of 13CH 3CO 2-Na and CH313C02-Na gave enough information to establish its structure. 相似文献
2.
The solution behavior of the single-stranded CCAAT-containing octamer 1 , d(AGCCAATA), that comprises part of the nuclear factor I (NF-I) recognition site at the origin of replication of human adenovirus has been studied by nmr spectroscopy at 500 and 600 MHz. Proton resonance assignments for 1 were aided by selective 13C enrichment at C1′ of A 1 or A 5. High-resolution 13C- 1H heteronuclear multiple-bond coherence spectra of the 13C-labeled oligomers permitted the selective detection of furanosyl ring protons within each labeled residue due to short- and long-range 13C- 1H couplings to the enriched C1′. The resulting assignments provided firm starting points in the interpretation of double quantum filtered correlated spectra, yielding information supplemented by total correlated spectroscopy (TOCSY) and rotating frame nuclear Overhauser effect spectroscopic data to completely assign the 1H-nmr spectrum of 1 and extract 3JHH values for furanose con-formational analysis. Several 13C- 1H spin-coupling constants within the 13C-enriched A 1 or A 5 residues were measured from cross-peak shifts in TOCSY spectra, and their signs determined by inspection of the relative orientations of these shifts. 1H- 2-H and 13C- 1H spin-couplings both indicate a preference (> 75%) for south (C2′- endo) conformations by the furanosyl rings of 1 . © 1994 John Wiley & Sons, Inc. 相似文献
3.
The synthesis of 1,3,5- 13C 3- and 2,4- 13C 2-labeled 5-O-bromobenzyl-2-deoxyribonolactones 2, precursors to 13C-enriched nucleoside phosphoramidites for solid-phase synthesis of DNA oligonucletides, is described. An equimolar combination of these two multiply labeled lactones affords a “population-labeled” mixture of isotopomers which exhibits an approximately 50-fold increase in the sensitivity of 13C-NMR compared to natural abundance measurements. The 13C- 13C 2-bond and 4-bond coupling constants are reported for the lactones; all are < 2 Hz, confirming that this labeling scheme should be especially useful for NMR-relaxation measurements. 相似文献
4.
Analysis of 2D [ 13C, 1H]-HSQC spectra of biosynthetic fractionally 13C labeled proteins is a reliable, straightforward means to obtain stereospecific assignments of Val and Leu methyl sites in proteins. Herein we show that the same fractionally labeled protein sample facilitates observation and identification of Phe and Tyr aromatic signals. This is the case, in part, because the fractional 13C labeling yields aromatic rings in which some of the 13C- 13C J-couplings, present in uniformly labeled samples, are absent. Also, the number of homonuclear J-coupling partners differs for the -, - and -carbons. This enabled us to vary their signal intensities in distinctly different ways by appropriately setting the 13C constant-time period in 2D [ 13C, 1H]-HSQC spectra. We illustrate the application of this approach to an 18 kDa protein, c-VIAF, a modulator of apoptosis. In addition, we show that cancellation of the aromatic 13C CSA and 13C- 1H dipolar interactions can be fruitfully utilized in the case of the fractionally labeled sample to obtain high resolution 13C constant-time spectra with good sensitivity. 相似文献
5.
The unloading of sucrose in the apical part of the hypocotyl of Ricinus communis L. seedlings was measured by 13C-nuclear magnetic resonance (NMR) spectroscopy. The cotyledons of the seedling were immersed in 5 mM Mes buffer containing
100 mM 13C-labeled sucrose. At intervals of 70–90 min, 13C-NMR spectra with broadband decoupling and nuclear Overhauser enhancement were acquired in vivo. The spectra showed growing 13C-resonances of the labeled positions in the sucrose molecule reaching steady-state labeling within 7–8 h. The specific 13C labeling of sucrose in the G 1-position changed from 0.38 in the supplied sucrose solution to 0.16 in the sucrose extracted from the hypocotyl piece at
the end of the experiment (13 h). Labeling of starch (and other insolubles) in the hypocotyl piece was ca. 0.10. It is proposed
that the decreased specific labeling of unloaded sucrose is mostly due to the separate local pools of sucrose in the cortex
and pith parenchyma, respectively, and less to continuous starch degradation and conversion to sucrose. The report gives an
example of the application of 13C-NMR spectroscopy in assimilate allocation studies.
Received: 10 October 1998 / Accepted: 31 December 1998 相似文献
6.
MAX8, a designer peptide known to undergo self-assembly following changes in temperature, pH, and ionic strength, has demonstrated usefulness for tissue engineering and drug delivery. It is hypothesized that the self-assembled MAX8 nanofiber structure consists of closed β-hairpins aligned into antiparallel β-sheets. Here, we report evidence from solid-state NMR spectroscopy that supports the presence of the hypothesized β-hairpin conformation within the nanofiber structure. Specifically, our 13C- 13C two-dimensional exchange data indicate spatial proximity between V3 and K17, and 13C- 13C dipolar coupling measurements reveal proximity between the V3 and V18 backbone carbonyls. Moreover, isotopic dilution of labeled MAX8 nanofibers did not result in a loss of the 13C- 13C dipolar couplings, showing that these couplings are primarily intramolecular. NMR spectra also indicate the existence of a minor conformation, which is discussed in terms of previously hypothesized nanofiber physical cross-linking and possible nanofiber polymorphism. 相似文献
7.
In annual crops, the partitioning of photosynthates to support root growth, respiration and rhizodeposition should be greater during early development than in later reproductive stages due to source/sink relationships in the plant. Therefore, seasonal fluctuations in carbon dioxide (CO 2) and nitrous oxide (N 2O) production from roots and root-associated soil may be related to resource partitioning by the crop. Greenhouse studies used 13C and 15N stable isotopes to evaluate the carbon (C) partitioning and nitrogen (N) uptake by corn and soybean. We also measured the CO 2 and N 2O production from planted pots as affected by crop phenology and N fertilization. Specific root-derived respiration was related to the 13C allocated to roots and was greatest during early vegetative growth. Root-derived respiration and rhizodeposition were greater for corn than soybean. The 15N uptake by corn increased between vegetative growth, tasseling and milk stages, but the 15N content in soybean was not affected by phenology. A peak in N 2O production was observed with corn at the milk stage, suggesting that the corn rhizosphere supported microbial communities that produced N 2O. Most of the 15N-NO 3 applied to soybean was not taken up by the plant and negative N 2O production during vegetative growth and floral initiation stages suggests that soybean roots supported the reduction of N 2O to dinitrogen (N 2). We conclude that crop phenology and soil N availability exert important controls on rhizosphere processes, leading to temporal variation in CO 2 and N 2O production. 相似文献
8.
Abstract Responses of whole body protein synthesis (WBPS) and glucose irreversible loss rate (ILR) were compared between dietary starch and sucrose in four male goats. Diets were fed at 1.2 times maintenance requirements of ME and CP with 30% of the ME as starch, starch plus sucrose or sucrose, twice daily. The diets consisted of 33, 32, 11 and 24% of alfalfa hay, corn, soybean meal and the carbohydrates, respectively. The WBPS and glucose ILR during 5 – 7 h after feeding were determined by an isotope dilution method of [ 2H 5]phenylalanine, [ 2H 2]tyrosine, [ 2H 4]tyrosine and [ 13C 6]glucose. Sucrose elevated ammonia nitrogen and lowered acetate concentrations in the rumen, but did not differ from starch in nitrogen retention. Glucose ILR and WBPS were similar between the carbohydrates. It was concluded that dietary sucrose would have effects similar to starch on WBPS and glucose kinetics in the absorptive state in goats fed a high-concentrate diet. 相似文献
9.
A simple spectral editing procedure is described that generates separate subspectra for the methyl 13C- 1H 3 multiplet components of 1H- 13C HSQC spectra. The editing procedure relies on co-addition of in-phase and antiphase spectra and yields 1H-coupled constant-time HSQC subspectra for the methyl region that have the simplicity of the regular decoupled CT-HSQC spectrum. Resulting spectra permit rapid and reliable measurement of 1H- 13C J and dipolar couplings. The editing procedure is illustrated for a Ca 2+-calmodulin sample in isotropic and liquid crystalline phases. 相似文献
10.
Fourier transform 13C nuclear magnetic resonance spectra have been obtained of intact, fresh soybean ovules ( Glycine max L. cv. Dare) harvested from pods subtended by a trifoliolate exposed to 13CO 2 1 to 3 days earlier. The high resolution spectra are interpreted in terms of the labeled sugars and lipids in the ovule. Comparison of the spectra taken over the 3-day period permits qualitative estimates of sugar metabolism and rates of lipid synthesis. The spectra also contain information about the distribution of labels within the lipid chains. This information leads to a method of estimating the extent to which glucose degradation in the synthesizing soybean ovule is involved in the reactions of the phosphogluconate pathway. 相似文献
11.
The kinetic behavior of translocation profiles indicates that their shape is determined largely by the rate at which tracer enters the sieve tubes in the source leaf. Confirmation of this relationship was sought by investigating the kinetics of 14C in the immediate source pool for translocated sucrose in soybean ( Glycine max L., cv. Bragg) and morning glory ( Ipomea nil Roth, cv. Scarlet O'Hara) leaves. Quantitative microautoradiography was used to follow the water-soluble 14C contents of the companion cells in minor veins after pulse-labeling with 14CO 2. In both morning glory and soybean, the observed kinetics in the companion cells matched reasonably well those expected from the shape of the translocation profiles. Marked compartmentation of sucrose was evident in soybean leaves in that the specific radioactivity of total leaf sucrose was greatest immediately after labeling and quickly declined, whereas labeling in the companion cells was low at first and did not reach a maximum for about 35 minutes. In morning glory leaves, the kinetics of sucrose specific radioactivity and of companion cell-labeling more closely paralleled one another. 相似文献
12.
13C- 13C NOESY experiments were performed under long mixing time conditions on reduced human superoxide dismutase (32 kDa, 15N, 13C and 70% 2H labeled). 13C- 13C couplings were successfully eliminated through post-processing of in-phase-anti-phase (IPAP) data. It appears that at mixing time m of 3.0 s the spin diffusion mechanism allows the detection of 96% of the two-bond correlations involving C and C . The interpretation was confirmed by simulations. This approach broadens the range of applicability of 13C- 13C NOESY spectroscopy. 相似文献
13.
The mechanisms of selective herbicidal action of sethoxydim were investigated by using cultured root tips of corn ( Zea mays L. cv Goldencrossbantam) and pea ( Pisum sativum L. cv Alaska). Meristematic cells in the cultured roots were arrested in G 1 and G 2 of the cell division cycle by sucrose starvation and resumed growth and cell division (proliferation) when sucrose was provided. Corn root growth after sucrose addition was inhibited by sethoxydim at concentrations of 0.01 micromolar and greater when roots were treated in the presence of sucrose but was not inhibited at 10 micromolar sethoxydim when they were treated during sucrose starvation. Greater absorption of [ 14C]sethoxydim into the meristematic region of corn roots was observed when cells were in proliferative condition but not when they were arrested by sucrose starvation, whereas no greater absorption of the herbicide into pea meristems was observed in either growth condition. In the cell cycle study, greater absorption of [ 14C]sethoxydim into the corn root meristem was observed at a certain limited time before S (DNA synthesis) stage. The physiological effects and the greater absorption of sethoxydim clearly depended on cell cycle progression of corn root meristem, whereas fatty acid synthesis, as well as its inhibition by sethoxydim, was not associated with either cell cycle progression or greater absorption of the herbicide. 相似文献
14.
Callus cultures derived from pith tissue of Nicotiana tabacum were grown on two media either under continuous illumination or in complete darkness. The first medium limited greening ability of callus grown in the light (3 milligrams per liter naphthalene acetic acid, 0.3 milligram per liter 2-isopentenylaminopurine, Murashige and Skoog salts, and 2% sucrose). The second medium encouraged chlorophyll synthesis (greening) though not shoot formation (0.3 milligram per liter naphthalene acetic acid; 0.3 milligrans per liter 2-isopentylaminopurine). To measure intracellular concentrations, calli were grown for 15 days on these standard media containing [U- 14C]sucrose. The dry weight proportions of the calli (as a fraction of fresh weight) and many metabolite concentrations nearly doubled in light-grown cells compared to dark-grown cells and increased 30 to 40% on low-auxin media relative to high-auxin media. Glutamine concentrations (from 4 to 26 millimolar) were very high, probably due to the NH 3 content of the media. Proline concentrations were 20-fold higher in calli grown on low-auxin media in the light (green cells), possibly a stress response to high osmotic potentials in these cells. To analyze sucrose metabolism, callus cells were allowed to take up 0.2% (weight per volume) [U- 14C]sucrose for up to 90 minutes. In callus tissues and in pith sections from stems of tobacco plants, sucrose was primarily metabolized through invertase activity, producing equal amounts of labeled glucose and fructose. Respiration of 14CO 2 followed the labeling patterns of tricarboxylic acid cycle intermediates. Photorespiration activity was low. 相似文献
15.
The complete 18C-NMR assignment of lysocellin sodium salt has been obtained based on the 13C- 13C double labeling method and comparison with its retroaldol degradation product as well as a structurally related polyether antibiotic, lasalocid A.In parallel, the biosynthesis of lysocellin was investigated by feeding 13C-labeled precursors followed by analyzing the resulting labeling patterns of the 13C-NMR spectra; a pathway to the antibiotic molecule is suggested which proceeds through condensation of two butyrate, eight propionate and one acetate units.In addition, a conversion of butyrate to propionate during the metabolic process has been disclosed. 相似文献
16.
Soybean ( Glycine max) yields high levels of both protein and oil, making it one of the most versatile and important crops in the world. Light has been implicated in the physiology of developing green seeds including soybeans but its roles are not quantitatively understood. We have determined the light levels reaching growing soybean embryos under field conditions and report detailed redox and energy balance analyses for them. Direct flux measurements and labeling patterns for multiple labeling experiments including [U‐ 13C 6]‐glucose, [U‐ 13C 5]‐glutamine, the combination of [U‐ 14C 12]‐sucrose + [U‐ 14C 6]‐glucose + [U‐ 14C 5]‐glutamine + [U‐ 14C 4]‐asparagine, or 14CO 2 labeling were performed at different light levels to give further insight into green embryo metabolism during seed filling and to develop and validate a flux map. Labeling patterns (protein amino acids, triacylglycerol fatty acids, starch, cell wall, protein glycan monomers, organic acids), uptake fluxes (glutamine, asparagine, sucrose, glucose), fluxes to biomass (protein amino acids, oil), and respiratory fluxes (CO 2, O 2) were established by a combination of gas chromatography‐mass spectrometry, 13C‐ and 1H‐NMR, scintillation counting, HPLC, gas chromatography‐flame ionization detection, C:N and amino acid analyses, and infrared gas analysis, yielding over 750 measurements of metabolism. Our results show: (i) that developing soybeans receive low but significant light levels that influence growth and metabolism; (ii) a role for light in generating ATP but not net reductant during seed filling; (iii) that flux through Rubisco contributes to carbon conversion efficiency through generation of 3‐phosphoglycerate; and (iv) a larger contribution of amino acid carbon to fatty acid synthesis than in other oilseeds analyzed to date. 相似文献
17.
Carbon-13 nuclear magnetic resonance (NMR) spectroscopy has been applied to the direct observation of acetate and pyruvate metabolism in suspension cultures of Zea mays (var Black Mexican Sweet). Growth of the corn cells in the presence of 2 millimolar [2- 13C]acetate resulted in a rapid uptake of the substrate from the medium and initial labeling (0-4 hours) of primarily the intracellular glutamate and malate pools. Further metabolism of these intermediates resulted in labeling of glutamine, aspartate, and alanine. With [1- 13C]acetate as the substrate very little incorporation into intermediary metabolites was observed in the 13C NMR spectra due to loss of the label as 13CO 2. Uptake of [3- 13C]pyruvate by the cells was considerably slower than with [2- 13C]acetate; however, the labelling patterns were similar with the exception of increased [3- 13C] alanine generation with pyruvate as the substrate. Growth of the cells for up to 96 hours with 2 millimolar [3- 13C]pyruvate ultimately resulted in labeling of valine, leucine, isoleucine, threonine, and the polyamine putrescine. 相似文献
18.
Methanobacterium espanolae, an acidiphilic methanogen, required acetate for maximal growth on H 2-CO 2. In the presence of 5 to 15 mM acetate, at a growth pH of 5.5, the μ max was 0.05 h -1. M. espanolae consumed 12.3 mM acetate during 96 h of incubation at 35°C with shaking at 100 rpm. At initial acetate levels of 2.5 to 10.0 mM, the amount of biomass produced was dependent on the amount of acetate in the medium. 13C nuclear magnetic resonance spectra of protein hydrolysates obtained from cultures grown on [1- 13C]- or [2- 13C]acetate indicated that an incomplete tricarboxylic acid pathway, operating in the reductive direction, was functional in this methanogen. The amino acids were labeled with a very high degree of specificity and at greater than 90% enrichment levels. Less than 2% label randomization occurred between positions primarily labeled from either the carboxyl or methyl group of acetate, and very little label was transferred to positions primarily labeled from CO 2. The labeling pattern of carbohydrates was typical for glucogenesis from pyruvate. This methanogen, by virtue of the properties described above and its ability to incorporate all of the available acetate (10 mM or lower) from the growth medium, has advantages over other microorganisms for use in the production of specifically labeled compounds. 相似文献
19.
Spectral editing is crucial to simplify the crowded solid-state NMR spectra of proteins. New techniques are introduced to edit 13C-13C correlations of uniformly labeled proteins under moderate magic-angle spinning (MAS), based on our recent frequency-selective homonuclear recoupling sequences [Zhang et al., J. Phys. Chem. Lett. 2020, 11, 8077–8083]. The signals of alanine, serine, or threonine residues are selected out by selective 13Cα-13Cβ double-quantum filtering (DQF). The 13Cα-13Cβ correlations of alanine residues are selectively established with efficiency up to?~?1.8 times that by dipolar-assisted rotational resonance (DARR). The techniques are shown in 2D/3D NCCX experiments and applied to the uniformly 13C, 15N labeled Aquaporin Z (AqpZ) membrane protein, demonstrating their potential to simplify spectral analyses in biological solid-state NMR. 相似文献
20.
The comprehensive structure determination of isotopically labeled proteins by solid-state NMR requires sequence-specific assignment of 13C and 15 N spectra. We describe several 2D and 3D MAS correlation techniques for resonance assignment and apply them, at 7.0 Tesla, to 13C and 15N labeled ubiquitin to examine the extent of resonance assignments in the solid state. Both interresidue and intraresidue assignments of the 13C and 15N resonances are addressed. The interresidue assignment was carried out by an N(CO)CA technique, which yields N i-C i–1 connectivities in protein backbones via two steps of dipolar-mediated coherence transfer. The intraresidue connectivities were obtained from a new 3D NCACB technique, which utilizes the well resolved C chemical shift to distinguish the different amino acids. Additional amino acid type assignment was provided by a 13C spin diffusion experiment, which exhibits 13C spin pairs as off-diagonal intensities in the 2D spectrum. To better resolve carbons with similar chemical shifts, we also performed a dipolar-mediated INADEQUATE experiment. By cross-referencing these spectra and exploiting the selective and extensive 13 C labeling approach, we assigned 25% of the amino acids in ubiquitin sequence-specifically and 47% of the residues to the amino acid types. The sensitivity and resolution of these experiments are evaluated, especially in the context of the selective and extensive 13C labeling approach. 相似文献
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