A method for the determination of changes of glycolytic metabolites in yeast on a subsecond time scale using extraction at neutral pH

Anal Biochem. 1992 Jul;204(1):118-23. doi: 10.1016/0003-2697(92)90149-2.

Abstract

Glucose metabolism in yeast can be stopped within 0.1 s by spraying the cells in 60% methanol at -40 degrees C. With this procedure the integrity of the cells is not damaged. Using stopped-flow equipment for the incubation with glucose, major changes within a second are shown to occur in intracellular glucose-6-phosphate whereas the fructose-1,6-bisphosphate concentration remains constant. After quenching, the cells can be separated from the medium, washed with cold methanol when required, and extracted using chloroform at -40 degrees C at neutral pH, ensuring minimal degradation of labile metabolites. With partly automated enzymatic methods, a large variety of metabolites, including all glycolytic intermediates, can be determined in the neutral extracts. During the first second after addition of glucose, a significant increase in free intracellular glucose is found.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Chloroform
  • Cold Temperature
  • Fructosediphosphates / metabolism
  • Glucose / metabolism
  • Glucose-6-Phosphate
  • Glucosephosphates / metabolism
  • Glycolysis*
  • Hydrogen-Ion Concentration
  • Intracellular Fluid / metabolism
  • Methanol
  • Methods
  • Saccharomyces cerevisiae / chemistry
  • Saccharomyces cerevisiae / metabolism*
  • Time Factors

Substances

  • Fructosediphosphates
  • Glucosephosphates
  • Glucose-6-Phosphate
  • Chloroform
  • Glucose
  • fructose-1,6-diphosphate
  • Methanol