Determination of intermediary metabolites in yeast. Critical examination of the effect of sampling conditions and recommendations for obtaining true levels

Abstract

The effect of sampling conditions on the levels of adenine nucleotides, pyridine nucleotides, glycolytic intermediates and related metabolites in yeast has been studied. A systematic examination of the conditions for harvesting has shown that it can be best accomplished by rapid filtration. Delays in the handling for removal of the medium, as is usual in the process of obtaining a number of data reported in the literature, lead to important changes in some of the metabolites examined. It is also shown that when a washing is imperative it can be carried out with a methanol-water mixture (50/50, v/v) cooled at −40° without loss of intracellular concentrations of non-readily diffusible metabolites. On the basis of this experience the outline of a generally applicable procedure is presented.