Evidence for monovalent phosphate transport in Ehrlich ascites tumor cells

In an effort to determine whether the Na + ‐dependent P i transport system of Ehrlich ascites tumor cells exhibits specificity for H 2 PO 4 − or HPO 4 −2 , P i fluxes were determined by measuring 32 P i P i self‐exchange. Three experimental approaches were employed. First, the effect of pH on steady‐state P i transport at 0.5 and 5 mM was studied. Second, the relationship between P i transport and P i concentration (0.25–9.2 mM) at pH 5.6 and 7.9 was determined. Third, the dependence of P i transport on [H 2 PO 4 − ] (0.05–4.2 mM) at constant [HPO 4 −2 ] (0.5 mM), and the converse, [HPO 4 −2 ] (0.06–4.5 mM) at constant [H 2 PO 4 − ] (0.5 mM), was evaluated. K s (apparent half‐saturation constant) and J max (maximal transport rate) were calculated by two methods: weighted linear regression (WLR) and a nonparametric procedure. The dependence of P i flux on pH indicates that optimum transport occurs at pH 6.9. P i transport decreases as pH is reduced when extracellular P i is either 0.5 or 5 mM. However, at pH 7.9, P i flux is reduced only in 0.5 mM P i . At pH 5.6, H 2 PO 4 − comprises 93% of the total P i present, and the calculated K s is 0.055 ± 0.026 mM (WLR). This is the same as the K s determined from the initial phase of the flux vs. [H 2 PO 4 − ] relationship (0.056 ± 0.020 mM). However, at pH 7.9 (where 94% of P i is HPO 4 −2 ), the measured K s is 0.58 ± 0.11 mM (WLR), which is ten times higher than at pH 5.6. This value is also five times greater than the K s calculated from the flux vs. [HPO 4 −2 ] curve (0.106 ± 0.16 mM). Kinetic parameters calculated by the nonparametric method, though somewhat different, gave similar relative results. Taken together, these results support two conclusions: (1) H 2 PO 4 − is the substrate for the Na + ‐dependent P i transport system of the Ehrlich cell, and (2) H + can inhibit P i transport.