Nucleoside translocation in sheep reticulocytes and fetal erythrocytes: a proposed model for the nucleoside transporter

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Abstract
Nucleoside transport by fetal erythrocytes from nucleoside-permeable and nucleoside-impermeable type newborn lambs and by reticulocytes from adult sheep was compared with that of mature erythrocytes from adult sheep of the 2 phenotypes. Fetal cells and reticulocytes transported [U(uniformly labeled)-14C]uridine rapidly, with little difference between cells from the 2 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 .apprx. 100-fold higher Vmax. This increased translocation capacity was associated with increased numbers of high-affinity [3H]nitrobenzylthioinosine binding sites (.apprx. 2000-3000 sites/cell vs. .apprx. 20 sites/cell for mature nucleoside-permeable sheep erythrocytes). The calculated transport capacity for each nucleoside translocation site was similar in all cell types (140-180 molecules/site/s at 25.degree. C, assuming that each transport site binds a single molecule of inhibitor). These values compared favorably with turnover estimates for the nucleoside transporter from human and pig erythrocytes. Loss of nucleoside transport activity after birth closely paralleled loss of [3H]nitrobenzylthioinosine binding sites and the progressive loss of fetal cells from the circulation. Reticulocyte maturation in vitro was also associated with rapid loss of both nucleoside transport capacity and inhibitor binding activity. p-Chloromercuriphenylsulfonate and trypsin had no effect on [3H]nitrobenzylthioinosine binding to intact fetal cells. Both agents markedly inhibited binding to isolated ghosts where both sides of the cell membrane were accessible to reagent. p-Chloromercuriphenylsulfonate inhibition was markedly reduced in the presence of uridine and reversed by addition of dithiothreitol. 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. The nucleoside carrier may exhibit chemical asymmetry. A simple molecular model of the erythrocyte nucleoside transporter consistent with these and other known properties of the carrier is proposed.