Nucleoside translocation in sheep reticulocytes and fetal erythrocytes: a proposed model for the nucleoside transporter.
1. Nucleoside transport by fetal erythrocytes from nucleoside-permeable and nucleoside-impermeable type new-born lambs and by reticulocytes from adult sheep was compared with that of mature erythrocytes from adult sheep of the two phenotypes.2. Fetal cells and reticulocytes transported [U-(14)C]uridine rapidly, with little difference between cells from the two types of sheep. Transport occurred by a saturable uptake mechanism with similar properties to that present in mature cells from adult nucleoside-permeable type animals, except for an approximately 100-fold higher V(max).3. This increased translocation capacity was associated with increased numbers of high-affinity [(3)H]nitrobenzylthioinosine binding sites ( approximately 2000-3000 sites/cell compared with approximately 20 sites/cell for mature nucleoside-permeable sheep erythrocytes).4. The calculated transport capacity for each nucleoside translocation site is therefore similar in all cell types (140-180 molecules/site. s at 25 degrees C, assuming that each transport site binds a single molecule of inhibitor). These values compare favourably with turnover estimates for the nucleoside transporter from human and pig erythrocytes.5. Loss of nucleoside transport activity after birth closely paralleled loss of [(3)H]nitrobenzylthioinosine binding sites and the progressive loss of fetal cells from the circulation. Similarly, reticulocyte maturation in vitro was also associated with rapid loss of both nucleoside transport capacity and inhibitor binding activity.6. p-Chloromercuriphenylsulphonate and trypsin had no effect on [(3)H]nitrobenzylthioinosine binding to intact fetal cells. In contrast, both agents markedly inhibited binding to isolated ;ghosts' where both sides of the cell membrane were accessible to reagent. p-Chloromercuriphenylsulphonate inhibition was markedly reduced in the presence of uridine, and reversed by addition of dithiothreitol.7. We conclude that nucleoside transport changes during ontogeny and reticulocyte maturation in the sheep as well as species differences in nucleoside transport capacity are regulated by variations in the numbers of functional transport sites per cell rather than by changes in the activity of a constant number of sites. It is also likely that the nucleoside carrier exhibits chemical asymmetry.8. A simple molecular model of the erythrocyte nucleoside transporter consistent with these and other known properties of the carrier is proposed.
D002731:4-Chloromercuribenzenesulfonate / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D001692:Biological Transport; D002463:Cell Membrane Permeability; D004912:Erythrocytes / Q000378:metabolism*; D005312:Fetal Blood / Q000378:metabolism*; D007700:Kinetics; D008954:Models, Biological; D009705:Nucleosides / Q000097:blood*; D012156:Reticulocytes / Q000378:metabolism*; D012756:Sheep; D013868:Thioinosine / Q000031:analogs & derivatives / Q000097:blood; D014357:Trypsin / Q000494:pharmacology; D014529:Uridine / Q000097:blood
D009705:Nucleosides; D013868:Thioinosine; D002731:4-Chloromercuribenzenesulfonate; D014357:Trypsin; C001789:4-nitrobenzylthioinosine; D014529:Uridine