A rapid and reliable method for metabolite extraction in yeast using boiling buffered ethanol

Authors

  • Benjamin Gonzalez,

    1. Laboratoire de Technologie de la Nutrition et de l'Alimentation, Clermont-Ferrand, France
    2. Centre de Bioingenierie Gilbert Durand, UMR-CNRS 5504, Laboratoire Associé INRA, Toulouse, France
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  • Jean François,

    Corresponding author
    1. Centre de Bioingenierie Gilbert Durand, UMR-CNRS 5504, Laboratoire Associé INRA, Toulouse, France
    • Département de Génie Biochimique et Alimentaire, Institut National des Sciences Appliquées, Complexe Scientifique de Rangueil, 31077 Toulouse Cedex 04, France.
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  • Michel Renaud

    1. Laboratoire de Technologie de la Nutrition et de l'Alimentation, Clermont-Ferrand, France
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Abstract

A simple and reliable method for the efficient inactivation of metabolism and for quantitative metabolite extraction from yeast cells is presented. It is based on the use of a boiling solution made of 75% ethanol (volume/final volume) buffered with 70 mm-Hepes (final concentration), pH 7·5, to guarantee the stability throughout the whole procedure of a large variety of metabolites, including all glycolytic intermediates, nucleotides, pyridine nucleotides and organic acids compounds. The extraction is fast, requiring only 3 min incubation of yeast cells in the ethanol-buffered mixture maintained at 80°C. It can be carried out either directly by spraying the cells into the boiling mixture, or after quenching the whole culture in 60% methanol kept at −40°C. Extracts are subsequently concentrated by evaporation under partial vacuum and the residue is resuspended in a small volume of water. This concentration step and the use of a highly sensitive analytical method allow us to quantify metabolites in less than 10 mg dry weight cells. This method, which can be applied to other fungi, could be very helpful for the determination of true metabolites in mutants generated through the EUROFAN programme and for metabolic flux analysis. © 1997 John Wiley & Sons, Ltd.

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