Separation of nitrobenzylthioinosine from high affinity binding sites on human erythrocytes by gel chromatography.
The potent inhibitor of nucleoside transport, 6-[(4-nitrobenzyl) mercapto]-9-beta-D-ribofuranosylpurine (NBMPR), binds reversibly, but with high affinity (KD 0.3-1.4 nM) to plasma membrane sites on human erythrocytes, and occupancy of those sites by NBMPR correlates with inhibition of transport. An earlier study from this laboratory showed that, upon photoactivation of site-bound [3H]NBMPR on erythrocyte membranes by exposure to UV light, isotopic ligand molecules became covalently linked to membrane polypeptides, which migrated as a discrete band on gel electrophoretograms, thereby identifying nucleoside transport polypeptides. The present study showed that erythrocytes on which the high affinity sites were occupied by reversibly bound [3H]NBMPR were freed of the latter by slow passage at 37 degrees C through 9 X 300 mm columns of Sephadex G-200 gel. Photoactivation of site-bound [3H]NBMPR on the intact cells caused apparent covalent attachment of the ligand because cells so treated retained the isotopic label during passage through the gel columns. Apparent covalent binding to erythrocytes also resulted from photoactivation of site-bound [3H]nitrobenzylmercaptopurine arabinoside.
D002850:Chromatography, Gel; D004912:Erythrocytes / Q000032:analysis*; D006801:Humans; D007288:Inosine / Q000031:analogs & derivatives*; D013868:Thioinosine / Q000031:analogs & derivatives* / Q000097:blood
D013868:Thioinosine; D007288:Inosine; C001789:4-nitrobenzylthioinosine