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.
Peerce
B E
BE
Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston 77550.
D000345:Affinity Labels; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives*; D005452:Fluoresceins / Q000494:pharmacology; D005456:Fluorescent Dyes; D007093:Imidazoles / Q000494:pharmacology; D007413:Intestinal Mucosa / Q000378:metabolism*; D008871:Microvilli / Q000378:metabolism; D010658:Phenylglyoxal / Q000031:analogs & derivatives / Q000494:pharmacology; D010710:Phosphates / Q000378:metabolism; D011817:Rabbits; D012964:Sodium / Q000494:pharmacology; D050608:Sodium-Phosphate Cotransporter Proteins; D013439:Sulfhydryl Reagents; D027981:Symporters; D013861:Thiocyanates / Q000494:pharmacology
D000345:Affinity Labels; D002352:Carrier Proteins; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007093:Imidazoles; D010710:Phosphates; D050608:Sodium-Phosphate Cotransporter Proteins; D013439:Sulfhydryl Reagents; D027981:Symporters; D013861:Thiocyanates; C059139:fluorescein isothiocyanate-phenylglyoxal; C059140:fluorescein n-acetylimidazole; D012964:Sodium; D016650:Fluorescein-5-isothiocyanate; D010658:Phenylglyoxal; C012160:N-acetylimidazole