Site-specific binding of nitrobenzylthioinosine (
NBMPR) to plasma membranes of some animal cells results in the inhibition of the facilitated diffusion of
nucleosides. The present study showed that
nucleoside transport in Novikoff UA rat
hepatoma cells is insensitive to site-saturating concentrations of
NBMPR. Equilibrium binding experiments demonstrated the presence of high-affinity sites for
NBMPR in a membrane-enriched fraction from these cells. In the presence of
uridine or
dipyridamole, specific binding of
NBMPR at these sites was inhibited. When Novikoff UA membranes were covalently labelled with [3H]
NBMPR by using photoaffinity techniques, specifically bound radioactivity was incorporated exclusively into a
polypeptide(s) with an apparent Mr of 72,000-80,000, determined by
sodium dodecyl sulphate/
polyacrylamide-gel electrophoresis. Covalent labelling of this
polypeptide was abolished in the presence of excess nitrobenzylthioguanosine (
NBTGR) and reduced in the presence of
adenosine,
uridine or
dipyridamole. The apparent Mr of the
NBMPR-binding
polypeptide in Novikoff UA cells is significantly higher than that reported for corresponding
polypeptides in other cell types (Mr 45,000-66,000). When membrane-enriched preparations from S49 mouse
lymphoma cells were photolabelled and mixed with labelled NovikoffUA membrane-enriched preparations, gel electrophoresis resolved the
NBMPR-binding
polypeptides from the two preparations.