Determination of de novo synthesized amino acids in cellular proteins revisited by 13C NMR spectroscopy
✍ Scribed by Ulrich Flögel; Wieland Willker; Dieter Leibfritz
- Publisher
- John Wiley and Sons
- Year
- 1997
- Tongue
- English
- Weight
- 336 KB
- Volume
- 10
- Category
- Article
- ISSN
- 0952-3480
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✦ Synopsis
C nuclear magnetic resonance spectroscopy was used to determine the absolute amounts of de novo synthesized amino acids in both the perchloric acid extracts and the hydrolyzed protein fractions of F98 glioma cells incubated for 2 h with 5 mmol/l [U-13 C]glucose. 13 C NMR spectra of the hydrolyzed protein fraction revealed a marked incorporation of 13 C-labelled alanine, aspartate and glutamate into the proteins of F98 cells within the incubation period. Additionally, small amounts of 13 C-labelled glycine, proline and serine could unambiguously be identified in the protein fraction. Astonishingly, approximately equal amounts of 13 C-labelled glutamate and aspartate were incorporated into the cellular proteins, although the cytosolic steady-state concentration of aspartate was below 13 C NMR detectability. Hypertonic stress decreased the incorporation of 13 C-labelled amino acids into the total protein, albeit their cytosolic concentrations were increased, which reflects an inhibition of protein synthesis under these conditions. On the other hand, hypotonic stress increased the amount of 13 Clabelled proline incorporated into the cellular proteins even though the cytosolic concentration of 13 C-labelled proline was largely decreased. Apparently, hypoosmotic conditions stimulate the synthesis of proteins or peptides with a high proline content. The results show that already after 2 h of incubation with [U-13 C]glucose there is a pronounced flux of 13 C label into the cellular proteins, which is usually disregarded if cytosolic fluids are examined only. This means that calculations of metabolic fluxes based on 13 C NMR spectroscopic data obtained from perchloric acid extracts of cells or tissues and also from in vivo measurements consider only the labelled 'NMR visible' cytosolic metabolites, which may have to be corrected for fast label flowing off into other compartments.