title: Phloretinyl-3'-benzylazide: a high affinity probe for the sugar transporter in human erythrocytes. I. Hexose transport inhibition and photolabeling of mutarotase.
abstract: A new phloretin derivative, phloretinyl-3'-benzylazide (PBAz), has been synthesized and compared with phloretin for its ability to inhibit the hexose transporter in human erythrocyte membranes in subdued light. Transport measurements were made using the light scattering (Orskov optical) method and a Millipore filtration technique with isotopically labeled sugars. Initial rates of sugar flux were measured under four different conditions to test for inhibition asymmetry. In each experimental condition, PBAz is from 6-20-times more potent than phloretin, making it one of the most effective reversible inhibitors known. Although both agents penetrate the cell membrane, they apparently fail to reach inhibitory levels at the inner surface over the time course of our nonequilibrated experiments, because of extensive binding to hemoglobin. The mechanism by which PBAz and its parent phloretin inhibit transport is pure competition with hexose for the carrier which faces the exterior of the membrane. If given time to equilibrate with the cells, the inhibition by both agents converts to a mixed type, i.e., both competitive and noncompetitive. The noncompetitive component could be due to inhibition of those transporter units oriented internally. Alternatively pre-equilibration with the inhibitors may cause them to attain high levels in the lipid membrane and produce nonspecific effects. PBAz and its precursor amine, phloretinyl-3'-benzylamine (PBA), compete with glucose for the sugar binding site on mutarotase at least as well as phloretin. When exposed to long wavelength ultraviolet radiation, PBAz is converted to a reactive intermediate which becomes covalently bound to the enzyme. Both irreversible ligand attachment and mutarotase inhibition are related to dose of the azide and irradiation time, but inactivation is from 5 to 6-times greater than label incorporation. We conclude that PBAz is a potentially useful photoaffinity labeling agent capable of covalently interacting with the transporter site facing the exterior of the red cell.
mesh_terms: D019325:3-O-Methylglucose; D001386:Azides / Q000494:pharmacology*; D001693:Biological Transport, Active / Q000187:drug effects; D002238:Carbohydrate Epimerases / Q000097:blood*; D002352:Carrier Proteins / Q000097:blood*; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism*; D006601:Hexoses / Q000097:blood*; D006801:Humans; D007700:Kinetics; D008757:Methylglucosides / Q000097:blood; D009004:Monosaccharide Transport Proteins; D010693:Phloretin / Q000494:pharmacology; D010782:Photolysis; D013329:Structure-Activity Relationship
chemical_list: D001386:Azides; D002352:Carrier Proteins; D006601:Hexoses; D008757:Methylglucosides; D009004:Monosaccharide Transport Proteins; D019325:3-O-Methylglucose; C033165:phloretinyl-3'-benzylazide; D002238:Carbohydrate Epimerases; C019646:aldose 1-epimerase; D010693:Phloretin