Identification of the intestinal Na-phosphate cotransporter

Na-dependent phosphate uptake in intestinal brush-border membrane vesicles was sensitive to arginine group-specific reagents in a substrate-sensitive manner. Four different arginine group-specific reagents were tested. All four reagents irreversibly inhibited Na-dependent phosphate uptake with the concentration for 50% inhibition, K0.5, varying between 150 and 40 microM. Maximum inhibition approached 80%. Addition of substrates during exposure to these reagents resulted in protection of Na-phosphate cotransport only in the presence of Na and phosphate. Na-phosphate cotransporter labeling at or near the phosphate site was accomplished using a pretreatment step with phenylglyoxal and substrates followed by a fluorescent phenylglyoxal-labeling step. Fluorescein isothiocyanate-phenylglyoxal (FITC-PG) specifically labeled a 130-kDa polypeptide on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in a substrate-sensitive manner, consistent with its effect on Na-phosphate cotransport. n-Acetylimidazole (NAI) inhibited Na-phosphate cotransport in a Na+- but not K+ -sensitive manner. NAI or fluorescein n-acetylimidazole (FNAI) inhibited Na-dependent phosphate uptake with a K0.5 for inhibition of 38 microM. Maximum inhibition of Na-phosphate cotransport was 75%. On SDS-PAGE, FNAI labeled five polypeptide bands in a Na-sensitive manner including the 130-kDa polypeptide band labeled by FITC-PG. Of these five bands only the 130-kDa polypeptide lost substrate protectability against FITC-PG inhibition of Na-phosphate cotransport and FITC-PG labeling on prior exposure to NAI in the absence of Na+. On this basis the 130-kDa polypeptide is tentatively identified as the intestinal Na-phosphate cotransporter and this polypeptide band contains both the Na substrate site and the phosphate substrate site.