Revised procedures for yeast metabolites extraction: application to a glucose pulse to carbon‐limited yeast cultures, which reveals a transient activation of the purine salvage pathway

In this study we have revised our original procedure of yeast metabolites extraction. We showed that: (a) less than 5% of intracellular metabolites leaks out during the step of rapid arrest of cellular metabolism by quenching yeast cells into a 60% methanol solution kept at −40 °C; and (b) with a few exception, the stability of metabolites were not altered during the 3 min boiling procedure in a buffered ethanol solution. However, there was a loss of external added metabolites of 5–30%, depending on the type of metabolites. This was mainly attributable to their retention on cellular debris after ethanol treatment, which prevented centrifugation of the cellular extracts before evaporation of ethanol. We further simplified our previous high‐performance ionic chromatography (HPIC) techniques for easier, more reliable and robust quantitative measurements of organic acids, sugar phosphates and sugar nucleotides, and extended these techniques to purine and pyrimidine bases, using a variable wavelength detector set at 220 and 260 nm in tandem with a pulsed electrochemical or suppressed conductivity detector. These protocols were successfully applied to a glucose pulse to carbon‐limited yeast cultures on purines metabolism. This study showed that glucose induced a fast activation of the purine salvage pathway, as indicated by a transient drop of ATP and ADP with a concomitant rise of IMP and inosine. This metabolic perturbation was accompanied by a rapid increase in the activity of the ISN1‐encoded specific IMP‐5′‐nucleotidase. The mechanism of this activation remains to be determined. Copyright © 2007 John Wiley & Sons, Ltd.

[1]  B. Palsson,et al.  The model organism as a system: integrating 'omics' data sets , 2006, Nature Reviews Molecular Cell Biology.

[2]  Jens Nielsen,et al.  The next wave in metabolome analysis. , 2005, Trends in biotechnology.

[3]  J. Nielsen,et al.  Global metabolite analysis of yeast: evaluation of sample preparation methods , 2005, Yeast.

[4]  Gregory Stephanopoulos,et al.  High-throughput metabolic state analysis: the missing link in integrated functional genomics of yeasts. , 2005, The Biochemical journal.

[5]  J. François,et al.  Acid trehalase in yeasts and filamentous fungi: localization, regulation and physiological function. , 2005, FEMS yeast research.

[6]  Christoph Wittmann,et al.  Dynamics of intracellular metabolites of glycolysis and TCA cycle during cell-cycle-related oscillation in Saccharomyces cerevisiae. , 2005, Biotechnology and bioengineering.

[7]  J. François,et al.  Two Distinct Pathways for Trehalose Assimilation in the Yeast Saccharomyces cerevisiae , 2004, Applied and Environmental Microbiology.

[8]  A. Sillero,et al.  In Saccharomyces cerevisiae, the effect of H2O2 on ATP, but not on glyceraldehyde-3-phosphate dehydrogenase, depends on the glucose concentration , 2004, Archives of Microbiology.

[9]  Mats Akesson,et al.  Simultaneous analysis of amino and nonamino organic acids as methyl chloroformate derivatives using gas chromatography-mass spectrometry. , 2003, Analytical biochemistry.

[10]  B. Daignan-Fornier,et al.  The yeast ISN1 (YOR155c) gene encodes a new type of IMP-specific 5'-nucleotidase , 2003, BMC Biochemistry.

[11]  A. Sillero,et al.  H2O2, but not menadione, provokes a decrease in the ATP and an increase in the inosine levels in Saccharomyces cerevisiae. An experimental and theoretical approach. , 2003, European journal of biochemistry.

[12]  Andrew Hayes,et al.  An optimized protocol for metabolome analysis in yeast using direct infusion electrospray mass spectrometry. , 2003, Phytochemistry.

[13]  T. Ferenci,et al.  Global metabolite analysis: the influence of extraction methodology on metabolome profiles of Escherichia coli. , 2003, Analytical biochemistry.

[14]  J. Heijnen,et al.  Rapid sampling for analysis of in vivo kinetics using the BioScope: a system for continuous-pulse experiments. , 2002, Biotechnology and bioengineering.

[15]  Christoph Wittmann,et al.  In vivo analysis of intracellular amino acid labelings by GC/MS. , 2002, Analytical biochemistry.

[16]  H. Lange,et al.  Analysis of glycolytic intermediates in Saccharomyces cerevisiae using anion exchange chromatography and electrospray ionization with tandem mass spectrometric detection , 2002 .

[17]  J J Heijnen,et al.  Improved rapid sampling for in vivo kinetics of intracellular metabolites in Saccharomyces cerevisiae. , 2001, Biotechnology and bioengineering.

[18]  E. Heinzle,et al.  Quantification of intracellular amino acids in batch cultures of Saccharomyces cerevisiae , 2001, Applied Microbiology and Biotechnology.

[19]  B. Daignan-Fornier,et al.  YLR209c Encodes Saccharomyces cerevisiae Purine Nucleoside Phosphorylase , 2001, Journal of bacteriology.

[20]  Oliver Fiehn,et al.  Combining Genomics, Metabolome Analysis, and Biochemical Modelling to Understand Metabolic Networks , 2001, Comparative and functional genomics.

[21]  J. François,et al.  Reserve carbohydrates metabolism in the yeast Saccharomyces cerevisiae. , 2001, FEMS microbiology reviews.

[22]  J. Heijnen,et al.  Glucose and the ATP paradox in yeast. , 2000, Biochemical Journal.

[23]  V. Hanko,et al.  Determination of carbohydrates, sugar alcohols, and glycols in cell cultures and fermentation broths using high-performance anion-exchange chromatography with pulsed amperometric detection. , 2000, Analytical biochemistry.

[24]  Ute Roessner,et al.  Simultaneous analysis of metabolites in potato tuber by gas chromatography-mass spectrometry. , 2000 .

[25]  François,et al.  Improved protocols for quantitative determination of metabolites from biological samples using high performance ionic-exchange chromatography with conductimetric and pulsed amperometric detection. , 2000, Enzyme and microbial technology.

[26]  Duboc,et al.  An interlaboratory comparison of physiological and genetic properties of four Saccharomyces cerevisiae strains. , 2000, Enzyme and microbial technology.

[27]  U. Roessner,et al.  Technical advance: simultaneous analysis of metabolites in potato tuber by gas chromatography-mass spectrometry. , 2000, The Plant journal : for cell and molecular biology.

[28]  L. Olsson,et al.  Cleanup and analysis of sugar phosphates in biological extracts by using solid-phase extraction and anion-exchange chromatography with pulsed amperometric detection. , 1998, Analytical biochemistry.

[29]  J. François,et al.  Sampling techniques and comparative extraction procedures for quantitative determination of intra- a , 1998 .

[30]  J. Boeke,et al.  Designer deletion strains derived from Saccharomyces cerevisiae S288C: A useful set of strains and plasmids for PCR‐mediated gene disruption and other applications , 1998, Yeast.

[31]  J. François,et al.  A rapid and reliable method for metabolite extraction in yeast using boiling buffered ethanol , 1997, Yeast.

[32]  B. Daignan-Fornier,et al.  The isolation and characterization of Saccharomyces cerevisiae mutants that constitutively express purine biosynthetic genes. , 1997, Genetics.

[33]  K. Nickerson,et al.  Single-run separation and detection of multiple metabolic intermediates by anion-exchange high-performance liquid chromatography and application to cell pool extracts prepared from Escherichia coli. , 1995, Analytical biochemistry.

[34]  M. Reuss,et al.  In vivo analysis of glucose-induced fast changes in yeast adenine nucleotide pool applying a rapid sampling technique. , 1993, Analytical biochemistry.

[35]  V. Schramm,et al.  Yeast AMP deaminase. Catalytic activity in Schizosaccharomyces pombe and chromosomal location in Saccharomyces cerevisiae. , 1993, The Journal of biological chemistry.

[36]  M. Konrad.  Identification and characterization of a yeast gene encoding an adenylate kinase homolog. , 1993, Biochimica et biophysica acta.

[37]  M. Deeley.  Adenine deaminase and adenine utilization in Saccharomyces cerevisiae , 1992, Journal of bacteriology.

[38]  K. van Dam,et al.  A method for the determination of changes of glycolytic metabolites in yeast on a subsecond time scale using extraction at neutral pH. , 1992, Analytical biochemistry.

[39]  J. Wild,et al.  Linear one-step assay for the determination of orthophosphate. , 1983, Analytical biochemistry.

[40]  E. Schaftingen,et al.  Fructose 2,6-bisphosphate. , 1982, Biochemical Society Transactions.

[41]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.