Effect of pH on the kinetics of Na +-dependent phosphate transport in rat renal brush-border membranes

Abstract

The kinetics of Na +-dependent phosphate uptake in rat renal brush-border membrane vesicles were studied under zero-trans conditions at 37°C and the effect of pH on the kinetic parameters was determined. When the pH was lowered it turned out to be increasingly difficult to estimate initial rates of phosphate uptake due to an increase in aspecific binding of phosphate to the brush border membrane. When EDTA or β-glycerophosphate was added to the uptake medium this aspecific binding was markedly reduced. At pH 6.8, initial rates of phosphate uptake were measured between 0.01 and 3.0 mM phosphate in the presence of 100 mM Na +. Kinetic analysis resulted in a non-linear Eadie-Hofstee plot, compatible with two modes of transport: one major low-affinity system ( K m ≈ 1.3 mM), high-capacity system ( V max ≈ 1.1 nmol/s per mg protein) and one minor high-affinity ( K m ≈ 0.03 mM), low-capacity system ( V max ≈ 0.04 nmol/s per mg protein). Na +-dependent phosphate uptake studied far from initial rate conditions i.e. at 15 s, frequently observed in the literature, led to a dramatic decrease in the V max of the low-affinity system. When both the extra- and intravesicular pH were increased from 6.2 to 8.5, the K m value of the low-affinity system increased, but when divalent phosphate is considered to be the sole substrate for the low-affinity system then the K m value is no longer pH dependent. In contrast, the K m value of the high-affinity system was not influenced by pH but the V max decreased dramatically when the pH is lowered from 8.5 to 6.2. These results suggest that the low-affinity, high-capacity system transports divalent phosphate only while the high-affinity, low-capacity system may transport univalent as well as divalent phosphate. Raising medium sodium concentration from 100 to 250 mM increased Na +-dependent phosphate uptake significantly but the pH dependence of the phosphate transport was not influenced. This observation makes it rather unlikely that pH changes only affect the Na + site of the Na +-dependent phosphate transport system.