Identification and Quantitation of Phosphorus Metabolites in Yeast Neutral pH Extracts by Nuclear Magnetic Resonance Spectroscopy

31P NMR spectroscopy offers a possibility to obtain a survey of all low-molecular-weight phosphorylated compounds in yeast. The yeast cells have been extracted using chloroform into a neutral aqueous phase. The use of high fields and the neutral pH extracts, which are suitable for NMR analysis, results in well-resolved 31P NMR spectra. Two-dimensional NMR experiments, such as proton-detected heteronuclear single quantum (1H-31P HSQC) and 31P correlation spectroscopy (31P COSY), have been used to assign the resonances. In the phosphomonoester region many of the signals could be assigned to known metabolites in the glycolytic and pentose phosphate pathways, although some signals remain unidentified. Accumulation of ribulose 5-phosphate, xylulose 5-phosphate, and ribose 5-phosphate was observed in a strain lacking transketolase activity when grown in synthetic complete medium. No such accumulation occurred when the cells were grown in yeast–peptone–dextrose medium. Trimetaphosphate (intracellular concentration about 0.2 mM) was detected in both cold methanol–chloroform and perchloric acid extracts.