title: External location of sites on pig erythrocyte membranes that bind nitrobenzylthioinosine.
abstract: Nucleoside transport in erythrocytes of various species is inhibited by the binding of nitrobenzylthioinosine (NBMPR) to high affinity sites associated with nucleoside transport elements of the plasma membrane. The present study examined binding of [3H]NBMPR to unsealed ghosts and to sealed right-side-out vesicles (ROVs) and inside-out vesicles (IOVs) prepared from pig erythrocytes. Kd values for NBMPR dissociation from the ligand-site complex in unsealed ghosts, ROVs and IOVs were similar (1.6-2.4 nM), and Bmax values (mean +/- SD) were, respectively, 22.2 +/- 5.5, 25.8 +/- 6.4, and 37.3 +/- 4.0 molecules/fg of protein, reflecting differences in the protein content of the membrane preparations. When temperatures were decreased from 22 degrees to 4 degrees, NBMPR binding to erythrocyte membrane preparations was reduced in IOVs relative to that in unsealed ghosts and ROVs. At 22 degrees, the association of NBMPR molecules with IOVs was slower than with ROVs and unsealed ghosts, differences that were virtually eliminated by permeabilization of the membrane preparations with saponin. Thus, the binding sites were more accessible to external NBMPR in sealed ROVs and unsealed ghosts than in sealed IOVs, indicating that the NBMPR sites are located on the extracellular aspect of the membrane.
authors.affiliation: McEachern Laboratory, University of Alberta, Edmonton, Canada.
authors.forename: F R
authors.initials: FR
authors.lastname: Agbanyo
authors.forename: C E
authors.initials: CE
authors.lastname: Cass
authors.forename: A R
authors.initials: AR
authors.lastname: Paterson
mesh_terms: D000818:Animals; D001665:Binding Sites; D001692:Biological Transport; D004910:Erythrocyte Membrane / Q000378:metabolism*; D066298:In Vitro Techniques; D007288:Inosine / Q000031:analogs & derivatives*; D009705:Nucleosides / Q000378:metabolism; D012503:Saponins / Q000494:pharmacology; D013552:Swine; D013868:Thioinosine / Q000031:analogs & derivatives* / Q000378:metabolism; D014316:Tritium
chemical_list: D009705:Nucleosides; D012503:Saponins; D014316:Tritium; D013868:Thioinosine; D007288:Inosine; C001789:4-nitrobenzylthioinosine