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°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°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. © 1992.