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+-dependent glucose transport in these membranes.
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D004396:Coloring Agents; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005456:Fluorescent Dyes; D005947:Glucose / Q000378:metabolism*; D007421:Intestine, Small / Q000378:metabolism*; D017879:Isothiocyanates; D007700:Kinetics; D008871:Microvilli / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins; D011487:Protein Conformation; D011817:Rabbits; D013861:Thiocyanates / Q000494:pharmacology
D002352:Carrier Proteins; D004396:Coloring Agents; D005452:Fluoresceins; D005456:Fluorescent Dyes; D017879:Isothiocyanates; D009004:Monosaccharide Transport Proteins; D013861:Thiocyanates; C005441:phenylisothiocyanate; D016650:Fluorescein-5-isothiocyanate; D005947:Glucose; C009941:potassium thiocyanate