A method for the determination of changes of glycolytic metabolites in yeast on a subsecond time scale using extraction at neutral pH.
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.
de Koning
W
W
E. C. Slater Institute for Biochemical Research, University of Amsterdam, The Netherlands.
van Dam
K
K
D002725:Chloroform; D003080:Cold Temperature; D005635:Fructosediphosphates / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D019298:Glucose-6-Phosphate; D005958:Glucosephosphates / Q000378:metabolism; D006019:Glycolysis; D006863:Hydrogen-Ion Concentration; D007424:Intracellular Fluid / Q000378:metabolism; D000432:Methanol; D008722:Methods; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000378:metabolism*; D013997:Time Factors
D005635:Fructosediphosphates; D005958:Glucosephosphates; D019298:Glucose-6-Phosphate; D002725:Chloroform; D005947:Glucose; C029063:fructose-1,6-diphosphate; D000432:Methanol