Use of 15N reverse gradient two-dimensional nuclear magnetic resonance spectroscopy to follow metabolic activity in Nicotiana plumbaginifolia cell-suspension cultures.
Nitrogen metabolism was monitored in suspension cultured cells of Nicotiana plumbaginifolia Viv. using nuclear magnetic resonance (NMR) spectroscopy following the feeding of (15NH4)2SO4 and K15NO3. By using two-dimensional 15N-1H NMR with heteronuclear single-quantum-coherence spectroscopy and heteronuclear multiple-bond-coherence spectroscopy sequences, an enhanced resolution of the incorporation of 15N label into a range of compounds could be detected. Thus, in addition to the amino acids normally observed in one-dimensional 15N NMR (glutamine, aspartate, alanine), several other amino acids could be resolved, notably serine, glycine and proline. Furthermore, it was found that the peak normally assigned to the non-protein amino-acid gamma-aminobutyric acid in the one-dimensional 15N NMR spectrum was resolved into a several components. A peak of N-acetylated compounds was resolved, probably composed of the intermediates in arginine biosynthesis, N-acetylglutamate and N-acetylornithine and, possibly, the intermediate of putrescine degradation into gamma-aminobutyric acid, N-acetylputrescine. The occurrence of 15N-label in agmatine and the low detection of labelled putrescine indicate that crucial intermediates of the pathway from glutamate to polyamines and/or the tobacco alkaloids could be monitored. For the first time, labelling of the peptide glutathione and of the nucleotide uridine could be seen.
Mesnard
F
F
Laboratoire de Biophysique, Groupe de Recherche des Biomolécules: micro-environment et Métabolisme, Faculté de Pharmacie, Amiens, France.
Azaroual
N
N
Marty
D
D
Fliniaux
M A
MA
Robins
R J
RJ
Vermeersch
G
G
Monti
J P
JP
D000596:Amino Acids / Q000378:metabolism*; D000645:Ammonium Sulfate / Q000378:metabolism; D002478:Cells, Cultured; D009682:Magnetic Resonance Spectroscopy; D009566:Nitrates / Q000378:metabolism; D009587:Nitrogen Isotopes; D010947:Plants, Toxic; D017680:Potassium Compounds / Q000378:metabolism; D014026:Nicotiana / Q000378:metabolism*; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
D000596:Amino Acids; D009566:Nitrates; D009587:Nitrogen Isotopes; D017680:Potassium Compounds; D005680:gamma-Aminobutyric Acid; C023844:potassium nitrate; D000645:Ammonium Sulfate