Exofacial photolabelling of the human erythrocyte glucose transporter with an azitrifluoroethylbenzoyl-substituted bismannose

Abstract

The synthesis of 2-N-[4-(1′-azitrifluoroethyl)benzoyl]-1,3-bis-(D-mannos-4-++ +yloxy)-2- propylamine (ATB-BMPA) is described. This compound was used as an exofacial probe for the human erythrocyte glucose-transport system. A new method is described for directly estimating the affinity for exofacial ligands which bind to the erythrocyte glucose transporter. By using this equilibrium-binding method, the Ki for ATB-BMPA was found to be 338 +/- 37 microM at 0 degrees C and 368 +/- 59 microM at 20 degrees C. This was similar to the concentration of ATB-BMPA required to half-maximally inhibit D-galactose uptake (Ki = 297 +/- 53 microM). The new photoaffinity reagent labelled the glucose transporter in intact cells but, because of its improved selectivity, was also used to label the glucose transporter in isolated erythrocyte membranes. The ATB-BMPA-labelled glucose transporter was 80% immunoprecipitated by anti-(GLUT1-C-terminal peptide) antibody, which shows that the GLUT1 glucose transporter is the major isoform present in erythrocytes. The labelling of the glucose transporter at its exofacial site, and the adoption of an outward-facing conformation, renders the transport system resistant to thermolysin and trypsin treatment. Trypsin treatment of the unlabelled glucose transporter in erythrocyte membranes produced an 18 kDa fragment which was subsequently labelled by ATB-BMPA, but had low affinity for this exofacial ligand. This suggests that the trypsin-treated transporter adopts an inward-facing conformation. The ability of D-glucose to displace ATB-BMPA from the native transporter and from the 18 kDa trypsin fragment have been compared. The D-glucose concentration which was required to obtain half-maximal inhibition of ATB-BMPA labelling was 6-fold lower for the 18 kDa tryptic fragment.