Nitrogen metabolism of asparagine and glutamate in Vero cells studied by H-1/N-15 NMR spectroscopy

摘要

Glutamine-free culture of Vero cells has previously been shown to cause higher cell yield and lower ammonia accumulation than that in glutamine-containing culture. Nitrogen metabolism of asparagine and glutamate as glutamine replacer was studied here using nuclear magnetic resonance (NMR) spectroscopy. N-15-labelled glutamate or asparagine was added and their incorporation into nitrogenous metabolites was monitored by heteronuclear multiple bond coherence (HMBC) NMR spectroscopy. In cells incubated with L-[N-15] glutamate, the N-15 label was subsequently found in a number of metabolites including alanine, aspartate, proline, and an unidentified compound. No detectable (NH4+)-N-15 signal occurred, indicating that glutamate was utilized by transamination rather than by oxidative deamination. In cells incubated with L-[2-N-15] asparagine, the N-15 label was subsequently found in aspartate, the amine group of glutamate/glutamine, and in two unidentified compounds. Incubation of cells with L-[4-N-15] asparagine showed that the amide nitrogen of asparagine was predominantly transferred to glutamine amide. There was no detectable production of (NH4+)-N-15, showing that most of the asparagine amide was transaminated by asparagine synthetase rather than deaminated by asparaginase. Comparing with a glutamine-containing culture, the activities of phosphate-activated glutaminase (PAG), glutamate dehydrogenase (GDH) and alanine aminotransferase (ALT) decreased significantly and the activity of aspartate aminotransferase (AST) decreased slightly.