Distance between substrate sites on the Na-glucose cotransporter by fluorescence energy transfer

Covalent fluorescent probes were used to label the rabbit intestinal brush border Na+-glucose cotransporter at the putative glucose and Na+ binding sites, and a steady-state fluorescence energy transfer technique was used to measure the distance between the two binding sites. In both intact brush border membrane vesicles and partially purified soluble protein, the distance (R2/3) between the Na+ and glucose sites was approximately equal to 35 A. This distance was the same with four different donor/acceptor pairs with different transfer efficiencies, by donor quantum yield measurements, or sensitized acceptor fluorescence. The fact that the Na+ site and glucose site probes bind to a 75-kDa polypeptide, copurify with the same isoelectric point (pI 5.3) and retain function, and exhibit energy transfer indicates that the sites are on the same 75-kDa polypeptide. The large distance between the Na+ and glucose site probes raises questions about simple models of frictional interactions between the two substrates during the transport cycle.