Reconstitution studies of the human erythrocyte nucleoside transporter.
The human erythrocyte nucleoside transporter has been identified as a band 4.5 polypeptide (Mr 45,000-66,000) on the basis of reversible binding and photoaffinity labeling experiments with the nucleoside transport inhibitor, nitrobenzylthioinosine (NBMPR). In the present study, the NBMPR-binding protein was extracted from protein-depleted human erythrocyte "ghosts" with Triton X-100 and reconstituted into soybean phospholipid vesicles by a freeze-thaw-sonication procedure. The reconstituted proteoliposomes exhibited nitrobenzylthioguanosine (NBTGR)-sensitive [14C]uridine transport. A partially purified preparation of the NBMPR-binding protein, consisting largely of band 4.5 polypeptides, was also shown to have nucleoside transport activity. This band 4.5 preparation exhibited a 10-fold increase in uridine transport activity and a 7-fold increase in NBMPR-binding activity relative to the crude membrane extract. Uridine transport by the reconstituted band 4.5 preparation was saturable (apparent Km = 0.21 mM; Vmax = 9 nmol/mg of protein/5 s) and was inhibited by dipyridamole, dilazep, adenosine, and inosine. The vesicles reconstituted with the band 4.5 preparation also exhibited stereospecific glucose transport which was inhibited by cytochalasin B, but unaffected by NBTGR. In contrast, cytochalasin B was a poor inhibitor of NBTGR-sensitive uridine transport. These experiments implicate band 4.5 polypeptides in both nucleoside and sugar permeation.
D001693:Biological Transport, Active / Q000187:drug effects; D001798:Blood Proteins / Q000032:analysis*; D004912:Erythrocytes / Q000032:analysis*; D005947:Glucose / Q000378:metabolism; D006151:Guanosine / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000032:analysis*; D008970:Molecular Weight; D033703:Nucleoside Transport Proteins; D017830:Octoxynol; D010743:Phospholipids / Q000378:metabolism; D010782:Photolysis; D011092:Polyethylene Glycols; D013010:Sonication; D013237:Stereoisomerism; D013868:Thioinosine / Q000031:analogs & derivatives / Q000378:metabolism; D013872:Thionucleosides / Q000378:metabolism; D013997:Time Factors; D014529:Uridine / Q000378:metabolism
D001798:Blood Proteins; D008565:Membrane Proteins; D033703:Nucleoside Transport Proteins; D010743:Phospholipids; D013872:Thionucleosides; D006151:Guanosine; C002121:6-(4-nitrobenzylthio)guanosine; D011092:Polyethylene Glycols; D013868:Thioinosine; D017830:Octoxynol; C001789:4-nitrobenzylthioinosine; D005947:Glucose; D014529:Uridine