Use of N-15 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 f ollowing the feeding of ((NH4)-N-15)(2)SO4 and (KNO3)-N-15 By using two-dim ensional N-15-H-1 NMR with heteronuclear single-quantum-coherence spectrosc opy and heteronuclear multiple-bond-coherence spectroscopy sequences, an en hanced resolution of the incorporation of N-15 label into a range of compou nds could be detected. Thus, in addition to the amino acids normally observ ed in one-dimensional N-15 NMR (glutamine, aspartate, alanine) several othe r amino acids could be resolved, notably serine, glycine and proline. Furth ermore, it was found that the peak normally assigned to the non-protein ami no-acid gamma-aminobutyric acid in the one-dimensional N-15 NMR spectrum wa s 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. Th e occurrence of N-15-label in agmatine and the low detection of labelled pu trescine indicate that crucial intermediates of the pathway from glutamate to polyamines and/or the tobacco alkaloids could be monitored. For the firs t time, labelling of the peptide glutathione and of the nucleotide uridine could be seen.