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Isabel Ayala Remy Sounier Nathalie Usé Pierre Gans Jérôme Boisbouvier 《Journal of biomolecular NMR》2009,43(2):111-119
A strategy for the introduction of (1H,13C-methyl)-alanine into perdeuterated proteins is described. Specific protonation of alanine methyl groups to a level of 95%
can be achieved by overexpressing proteins in M9/D2O based bacterial growth medium supplemented with 800 mg/l of 2-[2H], 3-[13C] l-alanine. However, though simple, this approach results in undesired, non-specific background labeling due to isotope scrambling
via different amino acid metabolic pathways. Following a careful analysis of known metabolic pathways we found that co-addition
of perdeuterated forms of α-ketoisovalerate-d7, succinate-d4 and l-isoleucine-d10 with labeled l-alanine, reduces undesired background labeling to <1%. When combined with recently developed methyl TROSY experiments, this
methyl-specific labeling protocol permits the acquisition of excellent quality correlation spectra of alanine methyl groups
in high molecular weight proteins. Our cost effective strategy offers a significant enhancement in the level of incorporation
of methyl-labeled alanine in overexpressed proteins over previously reported methods.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
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Dominic E. Mandy Joshua E. Goldford Hong Yang Doug K. Allen Igor G.L. Libourel 《The Plant journal : for cell and molecular biology》2014,77(3):476-486
13C metabolic flux analysis (MFA) has become the experimental method of choice to investigate the cellular metabolism of microbes, cell cultures and plant seeds. Conventional steady‐state MFA utilizes isotopic labeling measurements of amino acids obtained from protein hydrolysates. To retain spatial information in conventional steady‐state MFA, tissues or subcellular fractions must be dissected or biochemically purified. In contrast, peptides retain their identity in complex protein extracts, and may therefore be associated with a specific time of expression, tissue type and subcellular compartment. To enable ‘single‐sample’ spatially and temporally resolved steady‐state flux analysis, we investigated the suitability of peptide mass distributions (PMDs) as an alternative to amino acid label measurements. PMDs are the discrete convolution of the mass distributions of the constituent amino acids of a peptide. We investigated the requirements for the unique deconvolution of PMDs into amino acid mass distributions (AAMDs), the influence of peptide sequence length on parameter sensitivity, and how AAMD and flux estimates that are determined through deconvolution compare to estimates from a conventional GC–MS measurement‐based approach. Deconvolution of PMDs of the storage protein β–conglycinin of soybean (Glycine max) resulted in good AAMD and flux estimates if fluxes were directly fitted to PMDs. Unconstrained deconvolution resulted in inferior AAMD and flux estimates. PMD measurements do not include amino acid backbone fragments, which increase the information content in GC–MS‐derived analyses. Nonetheless, the resulting flux maps were of comparable quality due to the precision of Orbitrap quantification and the larger number of peptide measurements. 相似文献
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Tansley Review No. 22 What becomes of the transpiration stream? 总被引:2,自引:0,他引:2
M. J. CANNY 《The New phytologist》1990,114(3):341-368
359.
Transfusion tissue of pine needles as a site of retrieval of solutes from the transpiration stream 总被引:5,自引:2,他引:3
M. J. CANNY 《The New phytologist》1993,123(2):227-232
360.
Manuela Hartmann Mikhail V. Zubkov Adrian P. Martin David J. Scanlan & Peter H. Burkill 《FEMS microbiology letters》2009,298(2):166-173
Biologically available concentrations of individual dissolved amino acids in the open ocean are generally <1 nM. Despite this, the microbial turnover of amino acids is usually measured in hours indicating high demand. It is thought that the majority of uptake is due to bacterioplankton, although protists, particularly phototrophic protists, are also expected to take up amino acids. In order to assess the ability of protists to compete with prokaryotes for amino acids at subnanomolar concentrations, we examined the direct uptake of 3 H-leucine by phototrophic nanoflagellates (prasinophytes, pelagophytes and trebouxiophytes) and by associated bacteria using flow cytometric cell sorting. In contrast to 3 H-leucine-assimilating bacterial copopulations, none of the six studied nanoflagellates showed measurable direct uptake of 3 H-leucine, suggesting that the studied phototrophic protists were unable to utilize dissolved 3 H-leucine at natural oceanic concentrations. More practically, the flow-sorting technique allowed rapid and unequivocal differentiation of organic nitrogen uptake between prokaryotic cells and eukaryotic cells in mixed microbial populations, reducing the need to establish and maintain axenic algal cultures. 相似文献