Different molecular sizes for Na +-dependent phosphonoformic acid binding and phosphate transport in renal brush border membrane vesicles

Abstract

We compared several features of Na +-dependent phosphono[ 14C]formic acid (PFA) binding and Na +-dependent phosphate transport in rat renal brush border membrane vesicles. From kinetic analyses, we estimated an apparent K m for PFA binding of 0.86 mM, an order of magnitude greater than that for phosphate and the high-affinity phosphate transport system. A hyperbolic Na +-saturation curve for PFA binding and a sigmoidal Na +-saturation curve for phosphate transport were demonstrated; based on these data, we estimated stoichiometries of 1:1 for Na +/PFA and 2:1 for Na +/phosphate. By radiation inactivation analysis, target sizes for brush border membrane protein(s) mediating Na +-dependent PFA binding and Na +-dependent phosphate transport corresponded to molecular masses of 555 ± 32 kDa and 205 ± 36 kDa, respectively. Similar analysis of the phosphate-inhibitable component of Na +-dependent PFA binding gave a target size of 130 ± 28 kDa. We also demonstrated that phosphate deprivation, which elicits a 2.6-fold increase in brush border membrane Na +-dependent phosphate transport, had no effect on either Na +-dependent PFA binding or on the target size for PFA binding. However, phosphate deprivation appeared to increase the target size for phosphate transport (from 255 ± 32 to 335 ± 75 kDa ( P<0.01)). In summary, we present evidence for several differences between Na +-dependent PFA binding and Na +-dependent phosphate transport in rat renal brush border membrane vesicles and suggest that PFA may not interact exclusively with the proteins mediating Na +-phosphate co-transport.