Application of super(13)C-[2] - and super(13)C-[1,2] acetate in metabolic labelling studies of yeast and insect cells

The advantage of using super(13)C-labelled glucose in metabolic studies is that it is an important carbon and energy source for almost all biotechnologically and medically important organisms. On the other hand, the disadvantage is its relatively high cost in the labelling experiments. Looking for c...

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Bibliographic Details
Published in:Antonie van Leeuwenhoek 2006-05, Vol.89 (3-4), p.443-457
Main Authors: Paalme, Toomas, Nisamedtinov, Ildar, Abner, Kristo, Laht, Tiiu-Mai, Drews, Monika, Pehk, Tonis
Format: Article
Language:English
Subjects:
Acetic acid
Alanine
Amino acids
Carbon
Cell culture
Energy
Glucose
Glutamic acid
Insect cells
Malate dehydrogenase (oxaloacetate-decarboxylating) (NADP super(+))
Media (culture)
Metabolism
Minerals
Mitochondria
N.M.R
NADP
Saccharomyces uvarum
Spodoptera frugiperda
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Summary:The advantage of using super(13)C-labelled glucose in metabolic studies is that it is an important carbon and energy source for almost all biotechnologically and medically important organisms. On the other hand, the disadvantage is its relatively high cost in the labelling experiments. Looking for cheaper alternatives we found that super(13)C-[2] acetate or super(13)C-[1,2] acetate is a prospective compound for such experiments. Acetate is well incorporated by many organisms, including mammalian and insect cell cultures as preferred source of acetyl-CoA. Our experimental results using super(13)C NMR demonstrated that acetate was efficiently incorporated into glutamate and alanine secreted by the insect cell culture. Using D-stat culture of Saccharomyces uvarum on glucose/ super(13)C-acetate mineral media we demonstrated that the labelling patterns of proteinogenic amino acids can be well predicted on the basis of specific substrate consumption rates using the modified scheme of yeast metabolism and stoichiometric modelling. According to this scheme aspartate and alanine in S. uvarum under the experimental conditions used is synthesised in the mitochondria. Synthesis of alanine in the mitochondria was also demonstrated for Spodoptera frugiperda. For both organisms malic enzyme was also operative. For S. uvarum it was shown that the activity of malic enzyme is sufficient for supporting the mitochondrial biosynthetic reactions with NADPH.
ISSN:0003-6072
1572-9699
DOI:10.1007/s10482-005-9053-7