Exofacial photoaffinity labelling of the human erythrocyte sugar transporter.
The 4-azidosalicylate derivative of 1,3-bis(D-mannos-4'-yloxy)-2-[2-3H]propylamine (ASA-[2-3H]BMPA) has been tested as a photoaffinity label for the sugar transporter in human erythrocytes. When photolysed in the presence of intact erythrocytes, ASA-[2-3H]BMPA covalently binds to the exofacial surface of the transporter. This labelled protein appears as a broad band in the 4.5 region in sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis. The peak of radiolabel incorporation gives an apparent Mr of approx. 50 000 on 5-20% acrylamide gels. The binding is 80% inhibitable by 320 mM 4,6-O-ethylidene-D-glucose, by 320 mM D-glucose and by 50 microM cytochalasin B. Photoirradiation of a saturating concentration of ASA-BMPA in the presence of erythrocytes results in a 25-30% loss of D-galactose transport activity. From transport inactivation data and estimations of the amount of ASA-[2-3H]BMPA binding to the transporter it is calculated that there are approx. 220 000 exofacial hexose-transport binding sites per erythrocyte. The labelling of the transporter has been carried out using freshly drawn blood and 4-weeks-old transfusion blood. No change in the binding profile on SDS-polyacrylamide gel electrophoresis was observed. Proteolytic digestion of the ASA-[2-3H]BMPA-labelled transporter with either trypsin or alpha-chymotrypsin results in the appearance of a labelled 19 kDa fragment on SDS-polyacrylamide gel electrophoresis.
D000345:Affinity Labels / Q000378:metabolism*; D004187:Disaccharides / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D010777:Photochemistry
D000345:Affinity Labels; D004187:Disaccharides; D009004:Monosaccharide Transport Proteins; C044272:N-(4-azidosalicylamide)-1,2-bis(mannos-4'-yloxy)propyl-2-amine; D005947:Glucose