Research article

Studies of the intermediary metabolism in cultured cells of the insect Spodoptera frugiperda using ¹³C- or ¹⁵N-labelled tracers

Petra Adam¹ , Markus Gütlich¹ , Hartmut Oschkinat² , Adelbert Bacher¹ and Wolfgang Eisenreich¹

¹Lehrstuhl für Organische Chemie und Biochemie, Technische Universität München, Lichtenbergstr. 4, D-85747 Garching, Germany

²Forschungsinstitut für molekulare Pharmakologie, Robert-Rössle-Str. 10, D-13125 Berlin, Germany

author email corresponding author email

BMC Biochemistry 2005, 6:24doi:10.1186/1471-2091-6-24

Published:	14 November 2005

Abstract

Background

Insect cells can serve as host systems for the recombinant expression of eukaryotic proteins. Using this platform, the controlled expression of ¹⁵N/¹³C labelled proteins requires the analysis of incorporation paths and rates of isotope-labelled precursors present in the medium into amino acids. For this purpose, Spodoptera frugiperda cells were grown in a complex medium containing [U-¹³C₆]glucose. In a second experiment, cultures of S. frugiperda were grown in the presence of ¹⁵N-phenylalanine.

Results

Quantitative NMR analysis showed incorporation of the proffered [U-¹³C₆]glucose into the ribose moiety of ribonucleosides (40 – 45%) and into the amino acids, alanine (41%), glutamic acid/glutamine (C-4 and C-5, 30%) and aspartate/asparagine (15%). Other amino acids and the purine ring of nucleosides were not formed from exogenous glucose in significant amounts (> 5%). Prior to the incorporation into protein the proffered ¹⁵N-phenylalanine lost about 70% of its label by transamination and the labelled compound was not converted into tyrosine to a significant extent.

Conclusion

Growth of S. frugiperda cells in the presence of [U-¹³C₆]glucose is conducive to the fractional labelling of ribonucleosides, alanine, glutamic acid/glutamine and aspartic acid/asparagine. The isotopolog compositions of the ribonucleosides and of alanine indicate considerable recycling of carbohydrate intermediates in the reductive branch of the pentose phosphate pathway. The incorporation of ¹⁵N-labelled amino acids may be hampered by loss of the ¹⁵N-label by transamination.