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1.
High resolution structural elucidation of macromolecular structure by solid-state nuclear magnetic resonance requires the preparation of uniformly aligned samples that are isotopically labeled. In addition, to use the chemical shift interaction as a high resolution constraint requires an in situ tensor characterization for each site of interest. For 13C in the peptide backbone, this characterization is complicated by the presence of dipolar coupled 14N from the peptide bond. Here the 13C1-Gly2 site in gramicidin A is studied both as a dry powder and in a fully hydrated lipid bilayer environment. Linewidths reported for the oriented samples are a factor of five narrower than those reported elsewhere, and previous misinterpretations of the linewidths are corrected. The observed frequency from oriented samples is shown to be consistent with the recently determined structure for this site in the gramicidin backbone. It is also shown that, whereas a dipolar coupling between 13C and 14N is apparent in dry preparations of the polypeptide, in a hydrated bilayer the dipolar coupling is absent, presumably due to a `self-decoupling' mechanism. 相似文献
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J E Jentoft N Jentoft T A Gerken D G Dearborn 《The Journal of biological chemistry》1979,254(11):4366-4370
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In this paper, we analyzed the interaction of Z19 prolamin from a BR451 maize variety and pennisetin from a BRS1501 pearl millet variety with 1-(13)C-enriched oleic acid (OA) by (13)C NMR in solution. In both proteins, we identified the presence of free fatty acids by NMR in solid state and solution. The interactions were analyzed at the protein/OA molar ratios of 1:1 and 1:4. In the Z19/OA 1:1 mixture in 70% ethanol and 30% D(2)O, the chemical shift of OA C1 was 182.9 ppm, about 3 ppm above that of the pure OA in the same solvent. In contrast, upon addition of OA to the pennisetin (1:1), the chemical-shift value slightly decreased by less than 1 ppm. The chemical-shift titration curve of OA C1 in an apparent pH range of 5.5-7.3 shifted by approximately 0.3 pH units toward higher pH values in the pennisetin/OA 1:1 complex relative to the pure OA. The results obtained for the pennisetin/OA 1:4 mixture were similar to the complexes at a 1:1 molar ratio. A significant difference was observed between the 1:1 and 1:4 curves for Z19. The titration curve for Z19/OA 1:1 suggested specific binding at the sites with electrostatic interaction. 相似文献
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J J Ares J W Fowble T G Urchek D D Miller 《Biochemical and biophysical research communications》1987,142(3):1064-1071
Phenylisothiocyanate, enriched with 13C at the isothiocyanate carbon, has been synthesized and utilized as a 13C NMR probe of proteins for the first time. The reagent has been used to label the amino groups of oxidized glutathione, and the resulting 13C NMR spectrum shows a prominent thiocarbonyl peak after a single NMR scan. The reagent is also capable of differentiating amino groups on the insulin molecule with distinct peaks corresponding to the amino groups on the A and B chains of insulin. This study illustrates the potential of using a new 13C label to functionalize amino groups of proteins and to study the labeled proteins with 13C NMR. 相似文献
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The preparation of [1,2,3,4-13C] testosterone and of [1,2,3,4-13C] estradiol by total synthesis is described. The 13C labels are introduced by alkylating intermediate 1 with [1,2,3,4-13C]l-iodo-3,3-ethylenedioxybutane (2) to obtain intermediate 10. Hydrolysis of the ketal function, cyclization, aromatization and removal of protective groups gave [1,2,3,4-13C] estradiol. Labeled testosterone was prepared by methylating intermediate 10 and by subsequent treatment with acid. The labeled steroids can be used as tracers for in vivo metabolic studies and as internal standards for the development of definitive gc-ms quantitative methods. 相似文献
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Ernest Wenkert Hugo E. Gottlieb Otto R. Gottlieb Marilia O. da S. Pereira Mariza D. Formiga 《Phytochemistry》1976,15(10):1547-1551
The 13C NMR spectra of 15 neolignans of several structural types and two lignans were analyzed and their carbon shifts assigned. The shifts of pyrogallol ether and ethyl phenyl carbinyl ether models were used in this connection. The stereochemistry of a dimeric sideproduct in the preparation of the latter models was determined by 13C NMR analysis. 相似文献
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R C YashRoy 《Journal of biochemical and biophysical methods》1987,15(3-4):229-231
A novel approach to carbon-13 (13C) enrichment of chloroplast membranes (and plant materials in general) is presented for 13C-nuclear magnetic resonance (13C-NMR) studies. The method minimizes the occurrence of spectral complications arising from 13C-13C couplings resulting from a statistical distribution of 13C within the molecule with low probability of encountering two 13C atoms adjacent to each other. This is achieved by growing the plants in light surrounded by an atmosphere containing 1/3rd 12CO2 and 2/3rd 13CO2, liberated by weak acid-treatment of a mixture of corresponding barium carbonate salts. 相似文献
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The ribonuclease A derivative Npi-[13C1]carboxymethyl-histine-119 ribonuclease prepared by using [13C1]bromoacetate as alkylating reagent has been investigated with high resolution 13C NMR spectroscopy. In the 13C NMR spectra two carbon resonances of relatively high intensity appear which can be assigned to carboxyl groups attached to His-119 and Met-30, their intensity ratio being 10 : 1. The pH dependence of the carbon resonance of the carboxy-methyl group bound to the Npi of His-119 differs in the absence and presence of Cyd-2'-P, thus indicating that the catalytically inactive derivative does bind nucleotides. A mechanism of the alkylation reaction at pH 5.6 is proposed in which the epsilon-amino group of Lys-41 acts as the binding site for the carboxyl group of bromoacetate pushing the bromomethylene group towards the Npi of His-119 or the Ntau of His-12. 相似文献
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Plant and Soil - Plant-parasitic nematodes are able to sense and respond to gradients of chemical signals. How pH and inorganic salts in the rhizosphere affect nematode accumulation and... 相似文献
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Side chain dynamics monitored by 13C-13C cross-relaxation 总被引:1,自引:0,他引:1
A method to measure (13)C-(13)C cross-relaxation rates in a fully (13)C labeled protein has been developed that can give information about the mobility of side chains in proteins. The method makes use of the (H)CCH-NOESY pulse sequence and includes a suppression scheme for zero-quantum (ZQ) coherences that allows the extraction of initial rates from NOE buildup curves.The method has been used to measure (13)C-(13)C cross-relaxation rates in the 269-residue serine-protease PB92. We focused on C(alpha)-C(beta) cross-relaxation rates, which could be extracted for 64% of all residues, discarding serine residues because of imperfect ZQ suppression, and methyl (13)C-(13)C cross-relaxation rates, which could be extracted for 47% of the methyl containing C-C pairs. The C(alpha)-C(beta) cross-relaxation rates are on average larger in secondary structure elements as compared to loop regions, in agreement with the expected higher rigidity in these elements. The cross-relaxation rates for methyl containing C-C pairs show a general decrease of rates further into the side chain, indicating more flexibility with increasing separation from the main chain. In the case of leucine residues also long-range C(beta)-C(delta) cross-peaks are observed. Surprisingly, for most of the leucines a cross-peak with only one of the methyl C(delta) carbons is observed, which correlates well with the chi(2) torsion-angle and can be explained by a difference in mobility for the two methyl groups due to an anisotropic side chain motion. 相似文献
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[2-13C]Succinate has been used to examine the metabolic carbon flux from the Krebs cycle in rat renal proximal convoluted tubular (PCT) cells under physiological and pathophysiological conditions. Therefore, we developed a mathematical model that enabled us to determine the metabolic fluxes of the Krebs cycle. A mathematical model for the calculation of flux from [2-13C]succinate was used to determine fluxes in rat PCT cells during chronic acidosis in the presence and absence of 0.1 mM angiotensin II. The relative carbon efflux via glutamate dehydrogenase in rat renal PCT cells increases during chronic acidosis from 0.27 to 0.39, whereas this carbon flux is not affected by the presence of peptide hormone angiotensin II in the incubation medium. The fraction of intermediate 13C-labelled oxaloacetate transformed into the phosphoenolpyruvate and aspartate pools increases significantly from 0.41 to 0.57 in the case of chronic acidosis. The carbon efflux is not affected by angiotensin II. The 13C-NMR data also show that the carbon efflux through phosphoenolpyruvate carboxykinase increases from 0.35 to 0.56 in rat renal PCT cells derived from chronic acidotic animals, as well as in the presence of angiotensin II. The present results indicate that angiotensin II affects only the flux through phosphoenolcarboxykinase, whereas chronic acidosis increases the flux through phosphoenolpyruvate carboxykinase as well as the gluconeogenic flux. 相似文献
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L. G. Livingston Grace Medes With the Technical Assistance of Alice Shapiro 《The Journal of general physiology》1947,31(1):75-88
1. Starch, containing 7.05 atom per cent C13 excess has been produced in the mesophyll cells of bean leaves, starting with C13O2 containing 7.26 atom per cent C13 excess. Approximately 67 per cent of the C13 taken up by the leaves was determined in the starch fraction. 2. The soluble carbohydrate, containing 6.72 atom per cent C13 excess, accounts for approximately 23 per cent of the C13 taken up by the leaves. The remainder was principally in the coarse tissue fragments fraction (9.73 per cent of the C13 utilized). 3. The apparatus and procedures used in this experiment are described. 相似文献
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13C metabolic flux analysis. 总被引:8,自引:0,他引:8
W Wiechert 《Metabolic engineering》2001,3(3):195-206
Metabolic flux analysis using 13C-labeled substrates has become an important tool in metabolic engineering. It allows the detailed quantification of all intracellular fluxes in the central metabolism of a microorganism. The method has strongly evolved in recent years by the introduction of new experimental procedures, measurement techniques, and mathematical data evaluation methods. Many of these improvements require advanced skills in the application of nuclear magnetic resonance and mass spectrometry techniques on the one hand and computational and statistical experience on the other hand. This minireview summarizes these recent developments and sketches the major practical problems. An outlook to possible future developments concludes the text. 相似文献
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pH-dependence of 13C chemical shifts and 13C,H coupling constants in imidazole and L-histidine.
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The pH-dependence of selected 13C chemical shifts reflects the state of ionization of the imidazole ring in both imidazole and L-histidine. Titration of the amino and carboxyl groups of histidine also perturbs the shifts. The coupling constants 1J (13C(2),H) and 1J (13C(5),H) for both compounds also vary with pH, but in L-histidine these constants are relatively insensitive to the titration of groups outside the imidazole ring. 相似文献
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Discrimination between12C and13C by marine plants 总被引:2,自引:0,他引:2
Summary The natural abundance13C/12C ratios (as δ13C) of organic matter of marine macroalgae from Fife and Angus (East Scotland) were measured for comparison with the species'
ability to use CO2 and HCO
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for photosynthesis, as deduced from previously published pH-drift measurements. There was a clear difference in δ13C values for species able or unable to use HCO
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. Six species of Chlorophyta, 12 species of Phaeophyta and 8 species of Rhodophyta that the pH-drift data suggested could
use HCO
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had δ13C values in the range -8.81‰ to -22.55‰. A further 6 species of Rhodophyta which the pH-drift data suggested could only use
CO2 had δ13C values in the range -29.90‰ to-34.51‰. One of these six species (Lomentaria articulata) is intertidal; the other five are subtidal and so have no access to atmospheric CO2 to complicate the analysis. For these species, calculations based on the measured δ13C of the algae, the δ13C of CO2 in seawater, and the known13C/12C discrimination of CO2 diffusion and RUBISCO carboxylation suggest that only 15–21% of the limitation to photosynthesisin situ results from CO2 diffusion from the bulk medium to the plastids; the remaining 79–85% is associated with carboxylation reactions (and, via
feedback effects, down-stream processes). This analysis has been extended for one of these five species,Delesseria sanguinea, by incorporating data onin situ specific growth rates, respiratory rates measured in the laboratory, and applying Fick's law of diffusion to calculate a
boundary layer thickness of 17–24 μm. This value is reasonable for aDelesseria sanguinea frondin situ. For HCO
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-using marine macroalgae the range of δ13C values measured can be accommodated by a CO2 efflux from algal cells which range from 0.306 of the gross HCO
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influx forEnteromorpha intestinalis (δ13C=-8.81‰) in a rockpool to 0.787 forChondrus crispus (δ13C=-22.55‰). The relatively high computed CO2 efflux for those HCO
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-users with the more negative δ13C values implies a relatively high photon cost of C assimilation; the observed photon costs can be accommodated by assuming
coupled, energy-independent inorganic carbon influx and efflux. The observed δ13C values are also interpreted in terms of water movement regimes and obtaining CO2 from the atmosphere. Published δ13C values for freshwater macrophytes were compared with the ability of the species to use CO2 and HCO
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and again there was an apparent separation in δ13C values for these two groups. δ13C values obtained for marine macroalgae for which no pH-drift data are available permit predictions, as yet untested, as to
whether they use predominantly CO2 or HCO
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