Identification of the erythrocyte nucleoside transporter as a band 4.5 polypeptide. Photoaffinity labeling studies using nitrobenzylthioinosine.
Nitrobenzylthioinosine (NBMPR) was employed as a covalent probe of the erythrocyte nucleoside transporter. This nucleoside analogue, a potent inhibitor of nucleoside transport, binds tightly (KD = 10(-10) - 10(-9) M) but reversibly to specific sites on the carrier mechanism. High intensity UV irradiation of intact human erythrocytes, isolated "ghosts," and "protein-depleted" membranes in the presence of [3H]NBMPR and dithiothreitol (as a free radical scavenger) under nonequilibrium and equilibrium binding conditions resulted in selective covalent incorporation of 3H into the band 4.5 region of sodium dodecyl sulfate-polyacrylamide gels (Mr = 45,000-65,000). Covalent labeling of band 4.5 protein(s) under equilibrium binding conditions was inhibited by nitrobenzylthioguanosine, dipyridamole, uridine, and adenosine. A similar photolabeling pattern was observed using membranes from pig erythrocytes. In contrast, no incorporation of radioactivity into band 4.5 was observed under equilibrium binding conditions with membranes from nucleoside-impermeable sheep erythrocytes. These experiments suggest that the human and pig erythrocyte nucleoside transporters are band 4.5 polypeptides, a conclusion supported by previous isolation studies based on the assay of reversible [3H]NBMPR binding activity.
D000345:Affinity Labels; D001798:Blood Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002352:Carrier Proteins; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000378:metabolism*; D007288:Inosine / Q000031:analogs & derivatives*; D007700:Kinetics; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D008970:Molecular Weight; D033703:Nucleoside Transport Proteins; D010782:Photolysis; D013868:Thioinosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D014529:Uridine / Q000494:pharmacology
D000345:Affinity Labels; D001798:Blood Proteins; D002352:Carrier Proteins; D008565:Membrane Proteins; D033703:Nucleoside Transport Proteins; D013868:Thioinosine; D007288:Inosine; C001789:4-nitrobenzylthioinosine; D014529:Uridine