Bowen J W, Levinson C
J Cell Physiol. 1983 Aug;116(2):142-8. doi: 10.1002/jcp.1041160204.
In an effort to determine whether the Na+-dependent Pi transport system of Ehrlich ascites tumor cells exhibits specificity for H2PO4- or HPO4(-2), Pi fluxes were determined by measuring 32Pi-Pi self-exchange. Three experimental approaches were employed. First, the effect of pH on steady-state Pi transport at 0.5 and 5 mM was studied. Second, the relationship between Pi transport and Pi concentration (0.25-9.2 mM) at pH 5.6 and 7.9 was determined. Third, the dependence of Pi transport on [H2PO4-] (0.05-4.2 mM) at constant [HPO4(-2)] (0.5 mM), and the converse, [HPO4(-2)] (0.06-4.5 mM) at constant [H2PO4-] (0.5 mM), was evaluated. Ks (apparent half-saturation constant) and Jmax (maximal transport rate) were calculated by two methods: weighted linear regression (WLR) and a nonparametric procedure. The dependence of Pi flux on pH indicates that optimum transport occurs at pH 6.9. Pi transport decreases as pH is reduced when extracellular Pi is either 0.5 or 5 mM. However, at pH 7.9, Pi flux is reduced only in 0.5 mM Pi. At pH 5.6, H2PO4- comprises 93% of the total Pi present, and the calculated Ks is 0.055 +/- 0.026 mM (WLR). This is the same as the Ks determined from the initial phase of the flux vs. [H2PO4-] relationship (0.056 +/- 0.020 mM). However, at pH 7.9 (where 94% of Pi is HPO4(-2)), the measured Ks 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 Ks calculated from the flux vs. [HPO4(-20)] 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) H2PO4- is the substrate for the Na+-dependent Pi transport system of the Ehrlich cell, and (2) H+ can inhibit Pi transport.
为了确定艾氏腹水肿瘤细胞的钠依赖性磷酸根转运系统对H₂PO₄⁻ 还是HPO₄²⁻ 具有特异性，通过测量³²Pi-Pi自身交换来测定磷酸根通量。采用了三种实验方法。首先，研究了pH对0.5 mM和5 mM时稳态磷酸根转运的影响。其次，确定了pH 5.6和7.9时磷酸根转运与磷酸根浓度（0.25 - 9.2 mM）之间的关系。第三，评估了在恒定[HPO₄²⁻]（0.5 mM）下磷酸根转运对[H₂PO₄⁻]（0.05 - 4.2 mM）的依赖性，以及相反情况，即在恒定[H₂PO₄⁻]（0.5 mM）下对[HPO₄²⁻]（0.06 - 4.5 mM）的依赖性。通过两种方法计算Ks（表观半饱和常数）和Jmax（最大转运速率）：加权线性回归（WLR）和非参数程序。磷酸根通量对pH的依赖性表明，最佳转运发生在pH 6.9。当细胞外磷酸根为0.5 mM或5 mM时，随着pH降低，磷酸根转运减少。然而，在pH 7.9时，仅在0.5 mM磷酸根中磷酸根通量降低。在pH 5.6时，H₂PO₄⁻ 占总磷酸根的93%，计算得到的Ks为0.055±0.026 mM（WLR）。这与从通量与[H₂PO₄⁻]关系的初始阶段确定的Ks（0.056±0.020 mM）相同。然而，在pH 7.9（其中94%的磷酸根是HPO₄²⁻）时，测得的Ks为0.58±0.11 mM（WLR），比pH 5.6时高10倍。该值也比从通量与[HPO₄²⁻]曲线计算得到的Ks（0.106±0.16 mM）大5倍。通过非参数方法计算的动力学参数虽然有所不同，但给出了相似的相对结果。综上所述，这些结果支持两个结论：（1）H₂PO₄⁻ 是艾氏细胞钠依赖性磷酸根转运系统的底物，（2）H⁺ 可抑制磷酸根转运。
