Fluorescein isothiocyanate (FITC) was used to label the rabbit intestinal brush border Na+-glucose carrier, identify the carrier protein on sodium dodecyl sulfate/ polyacrylamide gel electrophoresis, and monitor the effect of ions and substrates on fluorescence quenching. Enriched brush border preparations were employed to study both glucose transport and FITC binding. FITC and a nonfluorescent analog (phenyl isothiocyanate, PITC) both inhibited Na+-dependent D-glucose transport irreversibly. Inhibition was blocked completely by the presence of Na+ and D-glucose during labeling. PITC was used to label nonspecific amino groups in the presence of glucose and Na+, and then the glucose carrier was labeled with FITC in the absence of substrates. Fluorescence of FITC bound to the carrier was quenched specifically with Na+ in a saturable fashion, and this indicates a Na+-dependent conformational change in the carrier. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of FITC-labeled membranes revealed specific labeling of a 71,000-dalton peptide. We conclude that Na+ induces a conformational shift in the 71,000-dalton glucose carrier, and this is quite consistent with the kinetics of Na+-dependent glucose transport in these membranes.Sodium-dependent organic solute transport across the brush border membrane of the small intestine plays a primary role in the absorption of sugars and amino acids (1). In these systems Na+ gradients provide the driving force for uphill solute uptake, and kinetic experiments suggest that Nat increases the affinity of the carrier for the solute through conformational changes. However, little direct information is available about either the nature of the conformation changes or even the identity of the carrier. Since site-specific reagents with fluorescent labels have been used successfully to probe conformation changes in other transport proteins (2-7), we have explored this approach with intestinal brush borders. Fluorescein isothiocyanate (FITC), an irreversible group-specific reagent for amino groups, has been used to label the Na+-glucose carrier, identify the carrier on NaDodSO4 gels, and monitor conformation changes. Nonspecific FITC binding was minimized by using enriched membrane preparations and labeling nonspecific sites with nonfluorescent isothiocyanates in the presence of excess Na+ and glucose. We find that Na+ produces a specific, saturable decrease in FITC fluorescence that is consistent with a Nat-dependent conformational change in the glucose camer. METHODSBrush border membrane vesicles (BBMVs) were isolated from rabbit small intestine by the Ca2t differential centrifugation method (8) and further purified by an adaptation of Hopfer's KSCN treatment (9). Briefly, the crude brush borders were resuspended by homogenization in 0.6 M KSCN and 10 mM Hepes/Tris buffer at pH 7.5, incubated at 40C, and then diluted 1:10 with 10 mM Hepes/Tris, pH 7.5-This suspension was centrifuged at 6000 x g for 10 min, and the resultant supernatant was centrifuged at 38,000 x g fo...

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.

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