Photoaffinity labeling of the human erythrocyte nucleoside transporter by N6-(p-Azidobenzyl)adenosine and nitrobenzylthioinosine. Evidence that the transporter is a band 4.5 polypeptide.
N6-(p-Azidobenzyl)adenosine (ABA) and nitrobenzylthioinosine (NBMPR) were employed as covalent probes of the nucleoside transport mechanism in human erythrocytes. NBMPR, a potent inhibitor of nucleoside transport, binds tightly (KD 0.3-1 nM) to specific sites on nucleoside transporter elements. ABA, a less potent inhibitor of uridine influx, competitively inhibited NBMPR binding (Ki 15 nM). [3H]ABA was bound tightly (KD 13.4 nM) but reversibly to sites on erythrocytes which appeared to be those which bind NBMPR. ABA binding was inhibited by uridine and adenosine. Irradiation with UV light caused site-bound [3H]ABA on erythrocyte membranes to become covalently bound and, similarly, photoactivation resulted in covalent attachment of membrane-bound [3H]NBMPR. In the presence of dithiothreitol, a free radical scavenger, photoactivation of the site-bound 3H-ligand on membranes depleted of extrinsic membrane proteins resulted in selective incorporation of 3H into band 4.5 of the membrane polypeptides which were resolved on sodium dodecyl sulfate-polyacrylamide gel electropherograms. This result, when considered with previous findings, indicates that the NBMPR-binding component of the nucleoside transport mechanism (or the entire mechanism, if the NBMPR site is an integral part) is a band 4.5 polypeptide.
D000345:Affinity Labels; D001386:Azides; D004229:Dithiothreitol; D004910:Erythrocyte Membrane / Q000032:analysis; D004912:Erythrocytes / Q000378:metabolism; D006801:Humans; D007288:Inosine / Q000031:analogs & derivatives*; D009705:Nucleosides / Q000378:metabolism*; D010777:Photochemistry; D013868:Thioinosine / Q000031:analogs & derivatives*; D013997:Time Factors; D014466:Ultraviolet Rays; D014529:Uridine / Q000097:blood
D000345:Affinity Labels; D001386:Azides; D009705:Nucleosides; D013868:Thioinosine; D007288:Inosine; C037241:N(6)-(4-azidobenzyl)adenosine; C001789:4-nitrobenzylthioinosine; D004229:Dithiothreitol; D014529:Uridine