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 degrees 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.
Loret
Marie Odile
MO
Laboratoire de Biotechnologie et Bioprocédés, UMR-CNRS 5504, UMR-INRA 792, Avenue de Rangueil, 31077 Toulouse Cedex 04, France.
Pedersen
Lene
L
François
Jean
J
D000227:Adenine Nucleotides / Q000032:analysis; D053000:Analytic Sample Preparation Methods; D005636:Fructosephosphates / Q000032:analysis; D005650:Fumarates / Q000032:analysis; D005947:Glucose / Q000378:metabolism*; D019298:Glucose-6-Phosphate / Q000032:analysis; D005958:Glucosephosphates / Q000032:analysis; D007288:Inosine / Q000032:analysis; D007291:Inosine Monophosphate / Q000032:analysis; D019289:Pyruvic Acid / Q000032:analysis; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000378:metabolism; D013403:Sugar Phosphates / Q000032:analysis; D014199:Trehalose / Q000031:analogs & derivatives / Q000032:analysis
D000227:Adenine Nucleotides; D005636:Fructosephosphates; D005650:Fumarates; D005958:Glucosephosphates; D013403:Sugar Phosphates; D007291:Inosine Monophosphate; C082722:trehalose-6-phosphate; D019298:Glucose-6-Phosphate; D007288:Inosine; C027618:fructose-6-phosphate; D019289:Pyruvic Acid; D014199:Trehalose; C031590:glucose-1-phosphate; D005947:Glucose