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. 1983 Dec 15;216(3):661–667. doi: 10.1042/bj2160661

Kinetics of nitrobenzylthioinosine binding to the human erythrocyte nucleoside transporter.

S M Jarvis, S N Janmohamed, J D Young
PMCID: PMC1152559  PMID: 6667262

Abstract

The kinetics of [3H]nitrobenzylthioinosine binding to human erythrocyte membranes was studied. The pseudo-first-order association was linear and consistent with a simple bimolecular reaction mechanism between nitrobenzylthioinosine and the nucleoside-transport mechanism. Dissociation of the [3H]nitrobenzylthioinosine complex at 22 degrees C was also linear (apparent k-1 congruent to 0.20 min-1). Adenosine was a competitive inhibitor of equilibrium high-affinity [3H]nitrobenzylthioinosine-binding activity (apparent Ki 0.1 mM). Dissociation of the [3H]nitrobenzylthioinosine-membrane complex was faster in the presence of adenosine and uridine, and this effect was proportional to the nucleoside concentration. Nucleoside concentrations less than 1 mM had no significant effect on the dissociation rate constant. In contrast, dissociation was slower in the presence of high concentrations (micromolar) of dipyridamole. Low concentrations of dipyridamole (2-200 nM) and nitrobenzylthioinosine concentrations as high as 2.5 microM had no effect on the rate of [3H]nitrobenzylthioinosine dissociation. These results are discussed in terms of possible distinct inhibitor and permeation sites, and are suggested to be consistent with both a single-site model for the binding of nitrobenzylthioinosine and permeant to the same site, or an allosteric-site model in which permeant and inhibitor bind to different sites.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Cass C. E., Dahlig E., Lau E. Y., Lynch T. P., Paterson A. R. Fluctuations in nucleoside uptake and binding of the inhibitor of nucleoside transport, nitrobenzylthioinosine, during the replication cycle of HeLa cells. Cancer Res. 1979 Apr;39(4):1245–1252. [PubMed] [Google Scholar]
  2. Cass C. E., Gaudette L. A., Paterson A. R. Mediated transport of nucleosides in human erythrocytes. Specific binding of the inhibitor nitrobenzylthioinosine to nucleoside transport sites in the erythrocyte membrane. Biochim Biophys Acta. 1974 Apr 12;345(1):1–10. doi: 10.1016/0005-2736(74)90239-9. [DOI] [PubMed] [Google Scholar]
  3. Cass C. E., Kolassa N., Uehara Y., Dahlig-Harley E., Harley E. R., Paterson A. R. Absence of binding sites for the transport inhibitor nitrobenzylthioinosine on nucleoside transport-deficient mouse lymphoma cells. Biochim Biophys Acta. 1981 Dec 21;649(3):769–777. doi: 10.1016/0005-2736(81)90182-6. [DOI] [PubMed] [Google Scholar]
  4. Cass C. E., Paterson A. R. Nitrobenzylthionionosine binding sites in the erythrocyte membrane. Biochim Biophys Acta. 1976 Jan 21;419(2):285–294. doi: 10.1016/0005-2736(76)90354-0. [DOI] [PubMed] [Google Scholar]
  5. DODGE J. T., MITCHELL C., HANAHAN D. J. The preparation and chemical characteristics of hemoglobin-free ghosts of human erythrocytes. Arch Biochem Biophys. 1963 Jan;100:119–130. doi: 10.1016/0003-9861(63)90042-0. [DOI] [PubMed] [Google Scholar]
  6. Eilam Y., Cabantchik Z. I. Nucleoside transport in mammalian cell membranes: a specific inhibitory mechanism of high affinity probes. J Cell Physiol. 1977 Aug;92(2):185–201. doi: 10.1002/jcp.1040920207. [DOI] [PubMed] [Google Scholar]
  7. Hammond J. R., Paterson A. R., Clanachan A. S. Benzodiazepine inhibition of site-specific binding of nitrobenzylthioinosine, an inhibitor of adenosine transport. Life Sci. 1981 Nov 23;29(21):2207–2214. doi: 10.1016/0024-3205(81)90492-6. [DOI] [PubMed] [Google Scholar]
  8. Harley E. R., Paterson A. R., Cass C. E. Initial rate kinetics of the transport of adenosine and 4-amino-7-(beta-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine (tubercidin) in cultured cells. Cancer Res. 1982 Apr;42(4):1289–1295. [PubMed] [Google Scholar]
  9. Jarvis S. M., Hammond J. R., Paterson A. R., Clanachan A. S. Nucleoside transport in human erythrocytes. A simple carrier with directional symmetry in fresh cells, but with directional asymmetry in cells from outdated blood. Biochem J. 1983 Feb 15;210(2):457–461. doi: 10.1042/bj2100457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Jarvis S. M., Hammond J. R., Paterson A. R., Clanachan A. S. Species differences in nucleoside transport. A study of uridine transport and nitrobenzylthioinosine binding by mammalian erythrocytes. Biochem J. 1982 Oct 15;208(1):83–88. doi: 10.1042/bj2080083. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Jarvis S. M., McBride D., Young J. D. Erythrocyte nucleoside transport: asymmetrical binding of nitrobenzylthioinosine to nucleoside permeation sites. J Physiol. 1982 Mar;324:31–46. doi: 10.1113/jphysiol.1982.sp014099. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Jarvis S. M., Young J. D., Ellory J. C. Nucleoside transport in human erythrocytes. Apparent molecular weight of the nitrobenzylthioinosine-binding complex estimated by radiation-inactivation analysis. Biochem J. 1980 Aug 15;190(2):373–376. doi: 10.1042/bj1900373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Jarvis S. M., Young J. D. Extraction and partial purification of the nucleoside-transport system from human erythrocytes based on the assay of nitrobenzylthioinosine-binding activity. Biochem J. 1981 Jan 15;194(1):331–339. doi: 10.1042/bj1940331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Jarvis S. M., Young J. D. Nucleoside translocation in sheep reticulocytes and fetal erythrocytes: a proposed model for the nucleoside transporter. J Physiol. 1982 Mar;324:47–66. doi: 10.1113/jphysiol.1982.sp014100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Jarvis S. M., Young J. D. Nucleoside transport in human and sheep erythrocytes. Evidence that nitrobenzylthioinosine binds specifically to functional nucleoside-transport sites. Biochem J. 1980 Aug 15;190(2):377–383. doi: 10.1042/bj1900377. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kolassa N., Jakobs E. S., Buzzell G. R., Paterson A. R. Manipulation of toxicity and tissue distribution of tubercidin in mice by nitrobenzylthioinosine 5'-monophosphate. Biochem Pharmacol. 1982 May 15;31(10):1863–1874. doi: 10.1016/0006-2952(82)90489-0. [DOI] [PubMed] [Google Scholar]
  17. Koren R., Cass C. E., Paterson A. R. The kinetics of dissociation of the inhibitor of nucleoside transport, nitrobenzylthioinosine, from the high-affinity binding sites of cultured hamster cells. Biochem J. 1983 Nov 15;216(2):299–308. doi: 10.1042/bj2160299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lynch T. P., Paran J. H., Paterson A. R. Therapy of mouse leukemia L1210 with combinations of nebularine and nitrobenzylthioinosine 5'-monophosphate. Cancer Res. 1981 Feb;41(2):560–565. [PubMed] [Google Scholar]
  19. Paterson A. R., Kolassa N., Cass C. E. Transport of nucleoside drugs in animal cells. Pharmacol Ther. 1981;12(3):515–536. doi: 10.1016/0163-7258(81)90096-6. [DOI] [PubMed] [Google Scholar]
  20. Paterson A. R., Lau E. Y., Dahlig E., Cass C. E. A common basis for inhibition of nucleoside transport by dipyridamole and nitrobenzylthioinosine? Mol Pharmacol. 1980 Jul;18(1):40–44. [PubMed] [Google Scholar]
  21. Plagemann P. G., Marz R., Wohlhueter R. M. Uridine transport in Novikoff rat hepatoma cells and other cell lines and its relationship to uridine phosphorylation and phosphorolysis. J Cell Physiol. 1978 Oct;97(1):49–72. doi: 10.1002/jcp.1040970107. [DOI] [PubMed] [Google Scholar]
  22. Wiley J. S., Jones S. P., Sawyer W. H., Paterson A. R. Cytosine arabinoside influx and nucleoside transport sites in acute leukemia. J Clin Invest. 1982 Feb;69(2):479–489. doi: 10.1172/JCI110472. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Wohlhueter R. M., Marz R., Plagemann P. G. Properties of the thymidine transport system of Chinese hamster ovary cells as probed by nitrobenzylthioinosine. J Membr Biol. 1978 Sep 19;42(3):247–264. doi: 10.1007/BF01870361. [DOI] [PubMed] [Google Scholar]
  24. Young J. D., Jarvis S. M. Nucleoside transport in animal cells. Biosci Rep. 1983 Apr;3(4):309–322. doi: 10.1007/BF01122895. [DOI] [PubMed] [Google Scholar]
  25. Young J. D., Jarvis S. M., Robins M. J., Paterson A. R. Photoaffinity labeling of the human erythrocyte nucleoside transporter by N6-(p-Azidobenzyl)adenosine and nitrobenzylthioinosine. Evidence that the transporter is a band 4.5 polypeptide. J Biol Chem. 1983 Feb 25;258(4):2202–2208. [PubMed] [Google Scholar]
  26. Young J. D. Nucleoside transport in sheep erythrocytes: genetically controlled transport variation and its influence on erythrocyte ATP concentrations. J Physiol. 1978 Apr;277:325–339. doi: 10.1113/jphysiol.1978.sp012274. [DOI] [PMC free article] [PubMed] [Google Scholar]

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