Incorporation of 32P into Phosphate Esters in Human Erythrocyte during Very Short Time Intervals

　As an attempt to elucidate the mechanism of phosphate uptake by human erythrocyte ³²P incorporation into acid-soluble organic phosphates during very short time periods of incubation with ³²P-orthophosphate was pursued by the use of ion exchange resin chromatography for the separation of the phosphorus compounds．Then seconds after addition of ³²P to the blood sample，only ATP and ADP were shown to be labelled with ³²P to a detectable extent, while the other substances, such as sugar phosphates and phosphoglycerates were not．The same results were obtained by incubation of the blood with ³²P for short time intervals of not more than 90 seconds．After 30 minutes，³²P was incorporated into sugar phosphates and phosphoglycerates．The results revealed that the incorporation of ³²P into 2, 3-diphosphoglycerate was a rather sluggish one．

　To obtain further information about the reaction sequence involved in the uptake of inorganic phosphate into organic phosphates inside the cells, the time course of ³²P-incorporation into ATP was determined．The radioactivity increased linearly with time．There appeared a short but distinct time lag before the increase strated．This suggests that a certain intermediary step may exist between inorganic orthophosphate in plasma and ATP in the cell．It is likely that the entry of phosphate involves phosphorylation during phosphoglyceraldehyde oxidation to 1, 3-diphosphoglycerate, localized at the cell surface．Unfortunately, the ester is extremely unstable so that separation and assay of this compound were impossible．There appeared highly radioactive unidentified phosphate compounds other than ATP and ADP，at the end of 10 seconds of incubation but none of them coincided with 1, 3-diphosphoglycerate．Furthermore, these compounds were often demonstrated in zerotime controls．Consequently too much biological significance should not be placed upon the presence of these compounds with radioactivity in very early periods of incubation．