INTRACELLULAR PHOSPHATE-WATER OXYGEN-EXCHANGE MEASURED BY MASS-SPECTROMETRY

To study, in vivo, potential P-i-water oxygen exchange catalyzed by ea ch of two high-affinity P-i symporters of Neurospora crassa, we have d eveloped methods for the purification of P-i from whole-cell extracts and the subsequent derivatization of P-i for analysis by GC-MS. We hav e also modified a published procedure for the preparation of O-18-P-i. However, the high background rate of transport-independent oxygen exc hange, determined by monitoring the appearance of O-18-P-i in cells in cubated in the presence of (H2O)-O-18, masks detection of any transpor t-dependent oxygen exchange which may occur. The rate of intracellular P-i-water oxygen exchange is 4.36 nmol O-18 incorporated into P-i per second per milligram of cell protein. The 180 isotopic distribution o f the intracellular P-i closely resembles that predicted for random ex change of a single P-i oxygen with that of water per enzymatic event. Based upon the observed isotopic enrichment, we calculate that the bul k intracellular P-i pool must undergo an average of one oxygen exchang e per phosphate ion about every 3 s. (C) 1996 Academic Press, Inc.