[125I]
iodohydroxynitrobenzylthioinosine ([125I]
IH-NBMPR), a new gamma-labeled
nucleoside transport inhibitor, has been prepared at a theoretical specific activity of 2000 Ci/mmol (1 Ci = 37 GBq).
IH-NBMPR was more acidic than hydroxynitrobenzylthioinosine (H-
NBMPR), having a pKa of 4.6. Site-specific binding of [125I]
IH-NBMPR to membrane-enriched fractions (MEF) from S49 mouse
lymphoma cells was pH dependent, increasing with the fraction of undissociated molecules present; it was maximal at pH 4.5 and negligible at pH 7.0. Scatchard analysis of specific binding to MEF from S49 cells under equilibrium conditions at pH 5.0 yielded a Kd of 15 nM (equivalent to 4.0 nM for the undissociated fraction of inhibitor molecules) and maximum number of binding sites (Bmax) of 4.9 pmol/mg
protein. Specific binding of
IH-NBMPR could not be demonstrated in MEF from AE1 cells, a
nucleoside transport-deficient mutant of S49 cells. Influx of
uridine into mouse erythrocytes at pH 5.0 in the presence of 5 microM
IH-NBMPR (1.4 microM undissociated
IH-NBMPR) was reduced to about 7% of the control value, indicating that this compound is an effective
nucleoside transport inhibitor. Photoactivation of site-bound [125I]
IH-NBMPR, following equilibration of the
ligand with MEF from S49 cells at pH 5.0, resulted in specific covalent labeling of a
polypeptide with a relative molecular mass of 52,000-63,000, identified on
sodium dodecyl sulfate-
polyacrylamide gels. These results indicate that the new, iodinated
ligand is an inhibitor of
nucleoside transport and that it binds specifically and with high affinity to
nucleoside transporter polypeptides in mammalian cells.