Erythrocyte nucleoside transport: asymmetrical binding of nitrobenzylthioinosine to nucleoside permeation sites

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Abstract
Nitrobenzylthioinosine is a potent and specific inhibitor of nucleoside translocation in animal cells. Kinetic and inhibitor binding studies were undertaken to clarify how this inhibitor interacts with the nucleoside transporter from human and nucleoside-permeable type sheep erythrocytes. [3H]nitrobenzylthioinosine inhibition of zero-trans [U(uniformly labeled)-14C]uridine influx into nucleoside-permeable type sheep cells was consistent with simple competitive inhibition (apparent Ki [inhibition constant] 1 nmol/l). Analysis of results using total inhibitor levels instead of cell-free inhibitor concentrations did not affect the inhibition pattern but increased the apparent Ki value by 5-fold. [3H]nitrobenzylthioinosine was a noncompetitive inhibitor of zero-trans[U-14C]uridine efflux (apparent Ki 1.5 nmol/l). Dipyridamole, another potent inhibitor of nucleoside translocation, also inhibited zero-trans[U-14C]uridine influx in a competitive manner (apparent Ki 20-40 nmol/l). [3H]nitrobenzylthioinosine bound to high-affinity sites on cell membranes from human and nucleoside-permeable type sheep cells (apparent Kd values .simeq. 1 nmol/l). Binding of inhibitor to these sites was competitively blocked by uridine, a well-characterized substrate for the nucleoside transporter (apparent Ki 1.25 and 0.9 mmol/l, respectively). These apparent Ki values were close to the apparent Km for uridine equilibrium exchange in human erythrocytes. Deoxycytidine was a competitive inhibitor of high-affinity [3H]nitrobenzylthioinosine binding activity (apparent Ki 1.0 and 1.2 mmol/l for human and nucleoside-permeable type sheep cell membranes, respectively). This contrasts with a previous report that this nucleoside had no effect on inhibitor binding activity. Transport studies confirmed that deoxycytidine is a substrate for the erythrocyte nucleoside transporter. Apparent Km and Vmax values for [U-14C]deoxycytidine zero-trans influx into human and nucleoside-permeable type sheep cells were comparable to those obtained for [U-14C]uridine. Nitrobenzylthioinosine competes directly with nucleosides for the permeation site of the nucleoside transporter, but inhibitor binds preferentially to the external membrane surface.

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