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


Journal of Microbial & Biochemical Technology

Author(s): de Koning W, van Dam K

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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.
This article was published in Anal Biochem and referenced in Journal of Microbial & Biochemical Technology

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