Re-examination of hexose-transporter inhibition and labelling by hexose isothiocyanates.
We have re-examined hexose-transport inhibition by hexose isothiocyanates and find that the inhibition is incomplete, probably because of decomposition of the reagent. The inhibition type is 'mixed', because hexose-transporter ligands such as maltose and cytochalasin B only give partial protection from inhibition. This suggests that a liganded-transporter-hexose isothiocyanate ternary complex is formed. We have compared the labelling of red-blood-cell membranes by [14C]MITC (D-maltose isothiocyanate) with the labelling obtained using a photoaffinity probe (ASA-BMPA [2-N-(4-azidosalicyloyl)-1,3-bis-(D-mannos-4'-yloxy)-2 -propylamine]) which gives specific labelling of the hexose transporter in band 4.5. [14C]MITC gives a partially D-glucose-displaceable labelling of a band 3 component in the same cell preparations which show ASA-BMPA labelling of band 4.5. This eliminates the possibility that band 4.5 labelling can only occur when the HITC (hexose isothiocyanate) binding protein in band 3 is proteolysed. HITC pretreatment does not decrease ASA-BMPA labelling of the exofacial site of band 4.5. This is also consistent with the formation of ternary complex. However, HITC pretreatment inhibits both reversible and photoactivated covalent [3H]cytochalasin B binding to band 4.5. These results suggest that, in the intact cell, interactions between a band 3 HITC-binding component and the inside cytochalasin B-binding site on the hexose transporter in band 4.5 may occur.
D000345:Affinity Labels / Q000494:pharmacology*; D001665:Binding Sites / Q000187:drug effects; D001693:Biological Transport, Active / Q000187:drug effects; D003571:Cytochalasin B / Q000378:metabolism; D004187:Disaccharides / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D006601:Hexoses / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D017879:Isothiocyanates; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000097:blood; D013861:Thiocyanates / Q000494:pharmacology*
D000345:Affinity Labels; D004187:Disaccharides; D006601:Hexoses; D017879:Isothiocyanates; D046911:Macromolecular Substances; D008565:Membrane Proteins; D013861:Thiocyanates; C052816:lactosyl isothiocyanate; D003571:Cytochalasin B; C024937:maltosyl isothiocyanate; C044272:N-(4-azidosalicylamide)-1,2-bis(mannos-4'-yloxy)propyl-2-amine; D005947:Glucose