Conformational Changes in the Intestinal Brush Border Sodium--Glucose Cotransporter Labeled with Fluorescein Isothiocyanate

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⁺-depedent glucose transport in these membranes.