Extraction and partial purification of the nucleoside-transport system from human erythrocytes based on the assay of nitrobenzylthioinosine-binding activity.
Nitrobenzylthioinosine, a potent nucleoside-transport inhibitor, binds to high-affinity sites on the human erythrocyte membrane. This binding is a specific interaction with functional nucleoside-transport sites. The protein(s) responsible for high-affinity nitrobenzylthioinosine binding was purified 13-fold by treatment of haemoglobin-free 'ghosts' with EDTA (pH 11.2) to remove extrinsic proteins, extraction of the protein-depleted membranes with Triton X-100 and passage of the soluble extract through a DEAE-cellulose column equilibrated with Triton X-100. Void-volume fractions were collected and treated with Bio-Beads SM-2 to remove detergent. These fractions contained 31% of the starting nitrobenzylthioinosine-binding activity. They also contained D-glucose-sensitive cytochalasin B-binding activity. Nitrobenzylthioinosine binding to the partially purified preparation was saturable (apparent Kd 1.6 nM) and inhibited by nitrobenzylthioguanosine, dipyridamole and uridine. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of pooled void-volume fractions revealed the presence of only two detectable protein bands, the broad zone 4.5 (containing glucose-transport protein) and a small amount of band 7.
D001692:Biological Transport / Q000187:drug effects; D002352:Carrier Proteins / Q000097:blood* / Q000302:isolation & purification; D002848:Chromatography, DEAE-Cellulose; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007288:Inosine / Q000031:analogs & derivatives*; D009705:Nucleosides / Q000097:blood*; D011092:Polyethylene Glycols / Q000494:pharmacology; D013868:Thioinosine / Q000031:analogs & derivatives* / Q000378:metabolism
D002352:Carrier Proteins; D009705:Nucleosides; D011092:Polyethylene Glycols; D013868:Thioinosine; D007288:Inosine; C001789:4-nitrobenzylthioinosine