Evidence for tyrosyl residues at the Na+ site on the intestinal Na+/glucose cotransporter.
A tyrosine group has been identified at, or near, the Na+-binding site of the Na+/glucose and Na+/proline cotransporters of rabbit intestinal brush-borders. Three tyrosine group-specific reagents, n-acetylimidazole, tetranitromethane, and p-nitrobenzene sulfonyl fluoride, were used to evaluate the role of tyrosyl groups in Na+-dependent glucose transport, Na+-dependent phlorizin binding, and the Na+-induced fluorescence quenching of fluorescein isothiocyanate bound to the glucose site of the carrier. All three reagents inhibited glucose transport, phlorizin binding, and fluorescein isothiocyanate quenching by 50-85% with Ki values in the range 7-50 microM. The presence of Na+ during the exposure of membranes to the reagents completely protected against inhibition, the Na+ concentration required to produce 50% protection was 14-36 mM. Fluorescent derivatives of n-acetylimidazole were synthesized to identify the tyrosyl residues on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A total of five polypeptide bands were labeled with eosin or fluorescein n-acetylimidazole in a Na+-sensitive manner. Two of these bands, previously identified as the glucose (75,000-dalton) and proline (100,000-dalton) binding sites of the glucose and proline carriers, account for 50% of the Na+-sensitive tyrosyl residues. On the basis of these studies, we believe that the Na+/glucose cotransporter contains both the Na+ and glucose active sites on the same polypeptide or that the cotransporter consists of two similar polypeptides, each containing one substrate binding site.
D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism*; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D007421:Intestine, Small / Q000378:metabolism*; D007700:Kinetics; D008871:Microvilli / Q000378:metabolism; D009004:Monosaccharide Transport Proteins; D010695:Phlorhizin / Q000378:metabolism; D011487:Protein Conformation / Q000187:drug effects; D011817:Rabbits; D012964:Sodium / Q000378:metabolism*; D013774:Tetranitromethane / Q000494:pharmacology; D013861:Thiocyanates / Q000378:metabolism; D014443:Tyrosine
D002352:Carrier Proteins; D005452:Fluoresceins; D009004:Monosaccharide Transport Proteins; D013861:Thiocyanates; D014443:Tyrosine; D012964:Sodium; D010695:Phlorhizin; D016650:Fluorescein-5-isothiocyanate; D005947:Glucose; D013774:Tetranitromethane