Photoaffinity labelling of nucleoside-transport proteins in plasma membranes isolated from rat and guinea-pig liver.
Nitrobenzylthioinosine (NBMPR) was employed as a probe of the nucleoside transporters from rat and guinea-pig liver. Purified liver plasma membranes prepared on self-generating Percoll density gradients exhibited 16-fold (rat) and 10-fold (guinea pig) higher [3H]NBMPR-binding activities than in crude liver homogenates (3.69 and 14.7 pmol/mg of protein for rat and guinea-pig liver membranes respectively, and 0.23 and 1.47 pmol/mg of protein for crude liver homogenates respectively). Binding to membranes from both species was saturable (apparent Kd 0.14 and 0.63 nM for rat and guinea-pig membranes respectively) and inhibited by uridine, adenosine, nitrobenzylthioguanosine (NBTGR) and dilazep. Uridine was an apparent competitive inhibitor of high-affinity NBMPR binding to rat membranes (apparent Ki 1.5 mM). There was a marked species difference with respect to dipyridamole inhibition of NBMPR binding (50% inhibition at 0.2 and greater than 100 microM for guinea-pig and rat respectively). These results are consistent with a role of NBMPR-binding proteins in liver nucleoside transport. Exposure of rat and guinea pig membranes to high-intensity u.v. light in the presence of [3H]NBMPR resulted in the selective radio-labelling of membrane proteins which migrated on sodium dodecyl sulphate/polyacrylamide gels with apparent Mr values in the same range as that of the human erythrocyte nucleoside transporter (45 000-66 000). Covalent labelling of these proteins was abolished when photolysis was performed in the presence of non-radio-active NBTGR as competing ligand.
D000345:Affinity Labels / Q000378:metabolism; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism; D004910:Erythrocyte Membrane / Q000378:metabolism; D006168:Guinea Pigs; D066298:In Vitro Techniques; D008099:Liver / Q000201:enzymology / Q000378:metabolism*; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism*; D009705:Nucleosides / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013868:Thioinosine / Q000031:analogs & derivatives / Q000378:metabolism; D014529:Uridine / Q000494:pharmacology
D000345:Affinity Labels; D002352:Carrier Proteins; D008565:Membrane Proteins; D009705:Nucleosides; D013868:Thioinosine; C001789:4-nitrobenzylthioinosine; D014529:Uridine