The use of methylphosphonate in the measurement of cytosolic pH in yeasts by 31P NMR

Abstract

At pH values of the cytosol far more higher than the pK of orthophosphate (Pi) the cytosolic pH cannot be determined accurately by means of 31P NMR from the chemical shift of Pi in the cell. In addition, in this pH range, resolution of Pi resonances is especially difficult because in highly energized cells internal Pi pools tend to be low. Under these conditions the pH of the cytosol of yeast can be determined more conveniently by means of 31P NMR of methylphosphonate loaded into the cells. This compound has a much higher pK than Pi and its cellular concentration is not reduced in metabolizing yeast cells. An additional advantage is that the cells can be loaded with relatively high amounts of this compound without affecting the energization of the cells allowing the determination of the cytosolic pH either at relatively low yeast densities and/or from spectra consisting of an appreciably reduced number of scans. In experiments in which N2 is bubbled through the yeast suspension in order to remove the CO2 liberated from the cells both the cytosolic pH determined by means of 31P NMR and the mean cell pH determined by means of a glass electrode after boiling the cells increase at the onset of metabolism. Addition of KCl leads to a further but transient increase in cytosolic pH. The difference between the cytosolic pH and the mean pH increases largely after the onset of metabolism pointing to an acidification of the vacuoles. Without N2 bubbling the cells become soon acidified after addition of glucose, which may be ascribed to the accumulation of bicarbonate in the cells. In this case the pH values determined after boiling the cells may be overestimated, because of volatilisation of the CO2 during the boiling procedure.