Intracellular Phosphate–Water Oxygen Exchange Measured by Mass Spectrometry

Abstract

To study,in vivo,potential Pi–water oxygen exchange catalyzed by each of two high-affinity Pisymporters ofNeurospora crassa,we have developed methods for the purification of Pifrom whole-cell extracts and the subsequent derivatization of Pifor analysis by GC–MS. We have also modified a published procedure for the preparation of18O-Pi. However, the high background rate of transport-independent oxygen exchange, determined by monitoring the appearance of18O-Piin cells incubated in the presence of H218O, masks detection of any transport-dependent oxygen exchange which may occur. The rate of intracellular Pi–water oxygen exchange is 4.36 nmol18O incorporated into Piper second per milligram of cell protein. The18O isotopic distribution of the intracellular Piclosely resembles that predicted for random exchange of a single Pioxygen with that of water per enzymatic event. Based upon the observed isotopic enrichment, we calculate that the bulk intracellular Pipool must undergo an average of one oxygen exchange per phosphate ion about every 3 s.