Determination of in-vivo cytoplasmic orthophosphate concentration in yeast

To investigate in-vivo concentrations of cytoplasmic phosphate, especially during dynamic conditions, a method has been developed that enables reproducible determination of cytoplasmic phosphate from 5 mu M up to 30 mu M. The method involves fast sampling. spontaneous inactivation of cell metabolism and differential extraction procedure to gain porosity of the outer cell membrane exclusively. To determine very low cytoplasmic phosphate concentrations, an enzymatic assay was linked to a sensitive spectrophotometric cycling method to increase the detection limit.