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J Physiol Vol 324 pp 31-46
Copyright © 1982 by The Physiological Society
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Erythrocyte nucleoside transport: asymmetrical binding of nitrobenzylthioinosine to nucleoside permeation sites

 S. M. Jarvis*, D. McBride{dagger} and J. D. Young{ddagger}

A.R.C. Institute of Animal Physiology, Babraham, Cambridge CB2 4AT

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. [3H]nitrobenzylthioinosine inhibition of zero-trans [U-14C]uridine influx into nucleoside-permeable type sheep cells was consistent with simple competitive inhibition (apparent Ki 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.

3. In contrast, [3H]nitrobenzylthioinosine was a non-competitive 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).

4. [3H]nitrobenzylthioinosine bound to high-affinity sites on cell membranes from human and nucleoside-permeable type sheep cells (apparent KD values ~= 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 are close to the apparent Km for uridine equilibrium exchange in human erythrocytes.

5. Similarly, deoxycytidine was found to be 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.

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.

* Present address: Cancer Research Unit, University of Alberta, Edmonton, Alberta, T6G 2H7, Canada.

{dagger} Present address: A.R.C. Animal Breeding Research Organization, The King's Buildings, West Mains Road, Edinburgh EH9 3JQ.

{ddagger} Present address: Department of Biochemistry, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong.




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