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A Method for the Determination of the Cellular Phosphorylation Potential and Glycolytic Intermediates in Yeast

✍ Scribed by P.G. Wallace; S.M. Pedler; J.C. Wallace; M.N. Berry

Publisher
Elsevier Science
Year
1994
Tongue
English
Weight
436 KB
Volume
222
Category
Article
ISSN
0003-2697

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✦ Synopsis

A method is described for rapidly quenching metabolism in yeast and extracting metabolites for analysis. The saponin digitonin is used to permeabilize the yeast cell membrane, in conjunction with perchloric acid (PCA) to quench metabolism and extract metabolites. Using this digitonin-PCA quench and extraction procedure, we have determined ATP concentrations in Saccharomyces cerevisiae in the range (2.89-3.39 \mathrm{mM}), [ATP]/[ADP] ratios of 4.5-6.6, and phosphorylation potentials of (48.7-49.9 \mathrm{~kJ} \cdot \mathrm{mol}^{-1}). A direct comparison with the currently accepted freeze/grind PCA/thaw procedure for extraction of yeast cell metabolites shows that essentially the same values are obtained by both techniques. The digitonin-PCA quench extraction method, used in conjunction with automated enzymatic analyses of metabolites, allows rapid extraction and analysis of large numbers of samples and metabolites and hence permits detailed investigations of intermediary metabolism in yeast. 1994 Academic Press, Inc.

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