Characterization of a photosensitive glucose derivative. A photoaffinity reagent for the erythrocyte hexose transporter.
The photosensitive reagent 6-N-(4-azido-2-hydroxy-3,5-diiodobenzoyl)-D-glucosamine has been assessed as a potential photoaffinity label for the hexose transporter. Under zero-trans conditions, transport experiments performed in the dark reveal that the reagent inhibits the uptake of D-glucose in resealed human erythrocyte ghosts. Increasing the concentration of glucose in the transport medium has a protective effect, reducing the inhibition. Kinetic analysis indicates that the probe acts as a competitive inhibitor with high affinity for the erythrocyte hexose transporter (Ki between 0.07 and 0.2 microM). Exposure to a 280 nm filtered high intensity mercury-vapor lamp results in a rapid and efficient photolysis. At low concentrations of the probe, specific labeling of membrane preparations was observed. Autoradiograms of 10% SDS gels revealed the specific labeling of bands 4.51 and 6. This labeling was concentration-dependent and protected by D-glucose (not the L-isomer) and phloretin in the medium. When subjected to multiple exposures of low concentration of the photoaffinity reagent, apparent saturation was achieved.
D000345:Affinity Labels; D001345:Autoradiography; D001386:Azides; D002352:Carrier Proteins / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000032:analysis*; D005944:Glucosamine / Q000031:analogs & derivatives*; D006801:Humans; D007700:Kinetics; D008433:Mathematics; D009004:Monosaccharide Transport Proteins; D010777:Photochemistry; D013053:Spectrophotometry
D000345:Affinity Labels; D001386:Azides; D002352:Carrier Proteins; D009004:Monosaccharide Transport Proteins; C044269:6-N-(4-azido-2-hydroxy-3,5-diiodobenzoyl)glucosamine; D005944:Glucosamine