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Erythrocyte nucleoside transport: asymmetrical binding of nitrobenzylthioinosine to nucleoside permeation sites
Author(s) -
Jarvis S. M.,
McBride D.,
Young J. D.
Publication year - 1982
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1113/jphysiol.1982.sp014099
Subject(s) - nucleoside , chemistry , permeation , erythrocyte membrane , biophysics , biochemistry , membrane , biology
1. 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. 2. [ 3 H]nitrobenzylthioinosine inhibition of zero‐ trans [U‐ 14 C]uridine influx into nucleoside‐permeable type sheep cells was consistent with simple competitive inhibition (apparent K i 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 K i value by 5‐fold. 3. In contrast, [ 3 H]nitrobenzylthioinosine was a non‐competitive inhibitor of zero‐ trans [U‐ 14 C]uridine efflux (apparent K i 1·5 nmol/l). Dipyridamole, another potent inhibitor of nucleoside translocation, also inhibited zero‐ trans [U‐ 14 C]uridine influx in a competitive manner (apparent K i 20‐40 nmol/l). 4. [ 3 H]nitrobenzylthioinosine bound to high‐affinity sites on cell membranes from human and nucleoside‐permeable type sheep cells (apparent K D values ≃ 1 nmol/l). Binding of inhibitor to these sites was competitively blocked by uridine, a well characterized substrate for the nucleoside transporter (apparent K i 1·25 and 0·9 mmol/l, respectively). These apparent K i values are close to the apparent K m for uridine equilibrium exchange in human erythrocytes. 5. Similarly, deoxycytidine was found to be a competitive inhibitor of high‐affinity [ 3 H]nitrobenzylthioinosine binding activity (apparent K i 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 K m and V max values for [U‐ 14 C]‐deoxycytidine zero‐ trans influx into human and nucleoside‐permeable type sheep cells were comparable to those obtained for [U‐ 14 C]uridine. 6. It is suggested from these results that nitrobenzylthioinosine competes directly with nucleosides for the permeation site of the nucleoside transporter, but that inhibitor binds preferentially to the external membrane surface.