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1.
Bi-directional translocation and degradation of Arginine (Arg) along the arbuscular mycorrhizal (AM) fungal mycelium were testified through 15N and/or 13C isotopic labeling. In vitro mycorrhizas of Glomus intraradices and Ri T-DNA-transformed carrot roots were grown in dual compartment Petri dishes. [15N- and/or13C]Arg was supplied to either the fungal compartment or the mycorrhizal compartment or separate dishes containing the uncolonized roots. The levels and labeling of free amino acids (AAs) in the mycorrhizal roots and in the extraradical mycelia(ERM) were measured by gas chromatography/mass spectrometry (GC-MS) and high-performance liquid chromatography (HPLC). The ERM of AM fungi exposed in either NH4 + or urea as sole external nitrogen source had much higher 15N enrichment of Arg, compared with those in nitrate or exogenous Arg; however, glycerol supplied as an external carbon source to the ERM had no significant effect on the level of Arg in the ERM. Meanwhile, Arg biosynthesized in the ERM could be translocated intact to the mycorrhizal roots and thereby the level of Arg in the mycorrhizal roots increased to about 20% after culture of ERM in 4 mmol/L NH4 + for 6 weeks. Also Arg was found to be bi-directionally transported along the AM fungal mycelium through [U-13C]Arg labeling either in the mycorrhizal compartment or in the fungal compartment. Once Arg was translocated to the potential N-limited sites, it would be further degraded into ornithine (Orn) and urea since either [U-13C] or [U-15N/U-13C]Orn was apparently shown up in the mycorrhizal root tissues when [U-13C] or [U-15N/U-13C]Arg was labeled in the fungal compartment, respectively. Evidently Orn formation indicated the ongoing activities of Arg translocation and degradation through the urea cycle in AM fungal mycelium. Supported by Science and Technology Department of Zhejiang Province (Grant No. 2006C22009).  相似文献   

2.
A simple labeling approach is presented based on protein expression in [1-13C]- or [2-13C]-glucose containing media that produces molecules enriched at methyl carbon positions or backbone Cα sites, respectively. All of the methyl groups, with the exception of Thr and Ile(δ1) are produced with isolated 13C spins (i.e., no 13C–13C one bond couplings), facilitating studies of dynamics through the use of spin-spin relaxation experiments without artifacts introduced by evolution due to large homonuclear scalar couplings. Carbon-α sites are labeled without concomitant labeling at Cβ positions for 17 of the common 20 amino acids and there are no cases for which 13Cα13CO spin pairs are observed. A large number of probes are thus available for the study of protein dynamics with the results obtained complimenting those from more traditional backbone 15N studies. The utility of the labeling is established by recording 13C R and CPMG-based experiments on a number of different protein systems.  相似文献   

3.
4.
The advantages of the organism Dictyostelium discoideum as an expression host for recombinant glycoproteins have been exploited for the production of an isotopically labeled cell surface protein for NMR structure studies. Growth medium containing [15N]NH4Cl and [13C]glycerol was used to generate isotopically labeled Escherichia coli, which was subsequently introduced to D. discoideum cells in simple Mes buffer. A variety of growth conditions were screened to establish minimal amounts of nitrogen and carbon metabolites for a cost-effective protocol. Following single-step purification by anion-exchange chromatography, 8 mg of uniformly 13C,15N-labeled protein secreted by approximately 1010D. discoideum cells was isolated from 3.3 liters of supernatant. Mass spectrometry showed the recombinant protein of 16 kDa to have incorporated greater than 99.9% isotopic label. The two-dimensional 1H-13C HSQC spectrum confirms 13C labeling of both glycan and amino acid residues of the glycoprotein. All heteronuclear NMR spectra showed a good dispersion of cross-peaks essential for high-quality structure determination.  相似文献   

5.
Direct uptake of organic nitrogen (ON) compounds, rather than inorganic N, by plant roots has been hypothesized to constitute a significant pathway for plant nutrition. The aim of this study was to test whether tomatoes (Solanum lycopersicum cv. Huying932) can take up ON directly from the soil by using 15NH4Cl, K15NO3, 1, 2-13C215N-glycine labeling techniques. The 13C and 15N in the plants increased significantly indicating that a portion of the glycine-N was taken up in the form of intact amino acids by the tomatoes within 48 h after injection into the soil. Regression analysis of excess 13C against excess 15N showed that approximately 21% of the supplied glycine-N was taken up intact by the tomatoes. Atom% excesses of 15N and 13C in the roots were higher than in any shoots. Results also indicated rapid turnover of amino acids (e.g., glycine) by soil microorganisms, and the poor competitive ability of tomatoes in absorbing amino acids from the soil solution. This implies that tomatoes can take up ON in an intact form from the soil despite the rapid turnover of organic N usually found under such conditions. Given the influence of climatic change and N pollution, further studies investigating the functional ecological implications of ON in horticultural ecosystems are warranted.  相似文献   

6.
It was attempted to examine whether or not isotope labeling may possibly affect an oligonuclear metal-thiolate cluster. Cu-metallothioneins are known to contain strongly distorted Cu-thiolate clusters and seemed appropriate for this study. Thus, yeast 13C-and 15N-Cu-metallothioneins were isolated from Saccharomyces cerevisiae cells grown in a minimal synthetic medium and some physicochemical parameters were compared with those of the unlabeled Cu-thionein. Surprisingly, the 13C- and 15N- labeled Cu7-thioneins are distinctly different in their characteristic spectroscopic properties. The electronic absorption was blue-shifted while both luminescence emission and chiroptic features display a distinct red shift with markedly diminished intensities, respectively. Contrary to common knowledge that isotope labeling does not affect the molecular architecture of a protein the present results support such a phenomenon. Attributable to the fortunate happenstance that there is a strongly distorted structural situation in the oligonuclear Cu-thiolate cluster this isotope effect came to light.  相似文献   

7.
Cost effective 13C/15N-isotope labeling of the avirulence protein AVR4 (10 kDa) of the fungal tomato pathogen Cladosporium fulvum was achieved with the methylotrophic yeast Pichia pastoris in a fermentor. The 13C/15N-labeled AVR4 protein accumulated to 30 mg/L within 48 h in an initial fermentation volume of only 300 mL, while prolonged optimized overexpressions yielded 126 mg/L. These protein yields were 24-fold higher in a fermentor than in flask cultures. In order to achieve these protein expression levels, we used the methanol-utilizing strain (Mut+) of Pichia pastoris which has a high growth rate while growing on methanol as the only carbon source. In contrast, the methanol-sensitive strain (MutS) could intrinsically yield comparable protein expression levels, but at the expense of additional carbon sources. Although both strains are generally used for heterologous protein expression, we show that the costs for 13C-isotope labeling can be substantially reduced using the Mut+ strain compared to the MutS strain, as no 13C3-glycerol is required during the methanol-induction phase. Finally, nitrogen limitations were precluded for 15N-labeling by an optimal supply of 10 g/L (15NH4)2SO4 every 24 h.  相似文献   

8.
Several techniques based on stable isotope labeling are used for quantitative MS. These include stable isotope metabolic labeling methods for cells in culture as well as live organisms with the assumption that the stable isotope has no effect on the proteome. Here, we investigate the 15N isotope effect on Escherichia coli cultures that were grown in either unlabeled (14N) or 15N‐labeled media by LC‐ESI‐MS/MS‐based relative protein quantification. Consistent protein expression level differences and altered growth rates were observed between 14N and 15N‐labeled cultures. Furthermore, targeted metabolite analyses revealed altered metabolite levels between 14N and 15N‐labeled bacteria. Our data demonstrate for the first time that the introduction of the 15N isotope affects protein and metabolite levels in E. coli and underline the importance of implementing controls for unbiased protein quantification using stable isotope labeling techniques.  相似文献   

9.
A simple isotope labeling approach for selective 13C/15N backbone labeling of proteins is described. Using {1,2-13C2}-pyruvate as the sole carbon source in bacterial growth media, selective incorporation of 13Cα-13CO spin-pairs into the backbones of protein molecules with medium-to-high levels of 13C-enrichment is possible for a subset of 12 amino acids. The isotope labeling scheme has been tested on a pair of proteins—a 7-kDa immunoglobulin binding domain B1 of streptococcal protein G and an 82-kDa enzyme malate synthase G. A number of protein NMR applications are expected to benefit from the {1,2-13C2}-pyruvate based protein production.  相似文献   

10.
A method is evaluated that employs variation in stable C and N isotopes from fractionations in C and N acquisition and growth to predict root biomasses of three plant species in mixtures. Celtis laevigata Willd. (C3), Prosopis glandulosa Torr. (C3, legume) and Schizachyrium scoparium (Michx.) Nash (C4), or Gossypium hirsutum L. (C3), Glycine max (L.) Merr. (C3 legume), and Sorghum bicolor (L.) Moench (C4) were grown together in separate, three-species combinations. Surface roots (0–10 cm depth) of each species from each of the two combinations were mixed in various proportions, and the relative abundances of 15N and 14N and 13C and 12C in prepared mixtures, surface roots of single species, and roots extracted from the 80-cm soil profile in which each species combination was grown were analyzed by mass spectrometry. An algebraic determination which employed the δ 13C, % 15N, and C and N concentrations of root subsamples of individual species accounted for more than 95% of the variance in biomass of each species in prepared mixtures with G. max, G. hirsutum, and S. bicolor. A similar analysis demonstrated species-specific differences in rooting patterns. Root biomasses of the C4 monocots in each combination, S. scoparium and S. bicolor, were concentrated in the upper 20 cm of soil, while those of G. hirsutum and the woody P. glandulosa were largest in lower soil strata. Analyses of stable C and N isotopes can effectively be used to distinguish roots of species which differ in ratios of 15N to 14N and 13C to 12C and thus to study belowground competition between or rooting patterns of associated species with different C and N isotope signatures. The method evaluated can be extended to quantify aboveground and belowground biomasses of component species in mixtures with isotopes of other elements or element concentrations that differ consistently among plants of interest.  相似文献   

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