Phloretinyl-3'-benzylazide: a high affinity probe for the sugar transporter in human erythrocytes. II. Irreversible transport inhibition is induced by photolysis.
In the dark, phloretinyl-3'-benzylazide (PBAz), at a nominal concentration of 10 microM, will inhibit the transport of D-glucose in human erythrocytes by more than 90%. This inhibition can be completely reversed by percolating the cell suspension through a small column of Sephadex G-10; cells recovered after this treatment, and then loaded with 100 mM D-glucose, possess a transport capacity (glucose efflux) equal to untreated cells. The Sephadex matrix completely removes non-covalently bound inhibitor even though, under these conditions (subdued light, 0.2% hematocrit, 0 degrees C, pH 6.2 or 7.8), from 70 to 80% of the PBAz added is bound to the cells (mostly non-specifically to hemoglobin). However, when erythrocytes exposed to 10 microM inhibitor are irradiated with long wavelength ultraviolet light, the glucose transporter is irreversibly inhibited; after 1 min irradiation, about 50% of transporter activity cannot be restored by Sephadex treatment. Under identical conditions, control cells (no PBAz, but irradiated and treated with Sephadex) retain over 90% of carrier activity. The photolytic conversion of the inhibition to an irreversible form is directly dependent on PBAz concentration. The results reaffirm our earlier conclusions that PBAz is a potentially useful photoaffinity labeling agent for the glucose transporter in erythrocyte membranes.
D000345:Affinity Labels / Q000494:pharmacology*; D001386:Azides / Q000494:pharmacology*; D001786:Blood Glucose / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D003624:Darkness; D004910:Erythrocyte Membrane / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D009004:Monosaccharide Transport Proteins; D010782:Photolysis
D000345:Affinity Labels; D001386:Azides; D001786:Blood Glucose; D002352:Carrier Proteins; D009004:Monosaccharide Transport Proteins; C033165:phloretinyl-3'-benzylazide