Intracellular phosphate--water oxygen exchange measured by mass spectrometry

To study, in vivo, potential P(i)-water oxygen exchange catalyzed by each of two high-affinity P(i) symporters of Neurospora crassa, we have developed methods for the purification of P(i) from whole-cell extracts and the subsequent derivatization of P(i) for analysis by GC-MS. We have also modified a published procedure for the preparation of 18O-P(i). However, the high background rate of transport-independent oxygen exchange, determined by monitoring the appearance of 18O-P(i) in cells incubated in the presence of H(2)18O, masks detection of any transport-dependent oxygen exchange which may occur. The rate of intracellular P(i)-water oxygen exchange is 4.36 nmol 18O incorporated into P(i) per second per milligram of cell protein. The 18O isotopic distribution of the intracellular P(i) closely resembles that predicted for random exchange of a single P(i) oxygen with that of water per enzymatic event. Based upon the observed isotopic enrichment, we calculate that the bulk intracellular P(i) pool must undergo an average of one oxygen exchange per phosphate ion about every 3 s.