Identification of the adenosine uptake sites in guinea pig brain.
Nitrobenzylthioinosine (NBMPR), a potent and specific inhibitor of nucleoside transport, was employed as a photolabile probe of the adenosine transporter in guinea pig brain membranes. Reversible, high-affinity binding of [3H]NBMPR to a crude preparation of guinea pig brain membranes was demonstrated (apparent KD 0.075 +/- 0.012 nM; Bmax values of 0.24 +/- 0.04 pmol/mg protein). Adenosine, uridine, dipyridamole, and nitrobenzylthioguanosine inhibited high-affinity binding. Low concentrations of cyclohexoadenosine (10-300 nM) had no effect on NBMPR binding. These properties of the high-affinity NBMPR binding sites were consistent with NBMPR binding to the nucleoside transport protein. Exposure of brain membranes in the presence of [3H]NBMPR and dithiothreitol, a free-radical scavenger, to ultraviolet light resulted in covalent incorporation of 3H into polypeptides of apparent MW 66,000-45,000, a value similar to that for the human erythrocyte nucleoside transporter. Covalent attachment of [3H]NBMPR was inhibited by adenosine, dipyridamole, and nitrobenzylthioguanosine.
D000241:Adenosine / Q000378:metabolism*; D000345:Affinity Labels / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000378:metabolism*; D004176:Dipyridamole / Q000494:pharmacology; D005260:Female; D006168:Guinea Pigs; D008566:Membranes / Q000378:metabolism; D008970:Molecular Weight; D010777:Photochemistry; D013868:Thioinosine / Q000031:analogs & derivatives / Q000378:metabolism
D000345:Affinity Labels; D013868:Thioinosine; D004176:Dipyridamole; C001789:4-nitrobenzylthioinosine; D000241:Adenosine