Binding of the Nucleoside Transport Inhibitor 4-Nitrobenzylthioinosine to Erythrocyte Membranes

Abstract

4-Nitrobenzylthioinosine (NBMPR), a potent nucleoside transport inhibitor, was prepared in two radioactive forms and the binding of these to erythrocyte ghosts was studied. Similar binding data were obtained with inhibitor containing 14 C in the purine 8-position or in the benzyl 7-position, suggesting that the entire inhibitor molecule was bound. A saturable high-affinity mode of NBMPR binding was apparent; NBMPR bound in this way was not removed by washing, but was displaced by a related inhibitor of nucleoside transport, 2-hydroxy-5-nitrobenzylthioguanosine (HNBTGR). It is postulated that the high-affinity binding sites are the nucleoside transport elements of the erythrocyte membrane. From ghosts treated with 14 C-NBMPR under conditions which assured binding of the high affinity type, 14 C was recovered by extractions in the form of NBMPR. Thus, this mode of NBMPR binding is reversible and covalent linkages do not appear to be involved. A low affinity mode of NBMPR binding was also demonstrated; this appeared to be a partition of NBMPR between the medium and the membrane substance. This component of bound NBMPR was not displaced by HNBTGR and was removed by washing.