Skip to main content
SpringerLink
Log in

Nucleoside transport in rat erythrocytes: two components with differences in sensitivity to inhibition by nitrobenzylthioinosine andp-chloromercuriphenyl sulfonate

  • Articles
  • Published:
The Journal of Membrane Biology Aims and scope Submit manuscript

Summary

The sensitivity of nucleoside transport by rat erythrocytes to inhibition by nitrobenzylthioinosine (NBMPR) and the slowly permeating organomercurial,p-chloromercuriphenyl sulfonate (pCMBS), was investigated. The dose response curve for the inhibition of uridine transport (100 μM) by NBMPR was biphasic −35% of the transport activity was inhibited with an IC50 value of 0.25 nM, but 65% of the activity remained insensitive to concentrations as high as 1 μM. These two components of uridine transport are defined as NBMPR-sensitive and NBMPR-insensitive, respectively. Uridine influx by both components was saturable and conformed to simple Michaelis-Menten kinetics, and was inhibited by other nucleosides. The uridine affinity of the NBMPR-sensitive transport component was threefold higher than for the NBMPR-insensitive transport mechanism (apparentK m for uridine 50±18 and 163±28 μM, respectively). The two transport systems also differed in their sensitivity topCMBS. NBMPR-insensitive uridine transport was inhibited bypCMBS with an IC50 of ∼25μM, while 1 mMpCMBS had little effect on NBMPR-sensitive transport by intact cells.pCMBS inhibition was reduced in the presence of uridine and adenosine and reversed by the addition by β-mercaptoethanol, suggesting that thepCMBS-sensitive thiol group is located on the exterior surface of the erythrocyte membrane within the nucleoside binding site of the transport system. Inhibition of uridine transport by NBMPR was associated with high-affinity [3H]NBMPR binding to the cell membrane (apparentK d46±25 pM). Binding of inhibitor to these sites was competitively blocked by uridine and inhibited by adenosine, thymidine, dipyridamole, dilazep and nitrobenzylthioguanosine. Assuming that each NBMPR-sensitive transport site binds a single molecule of NBMPR, the calculated translocation capacity of each site is 25±6 molecules/site per sec at 22°C.pCMBS had no effect on [3H]NBMPR binding to intact cells but markedly inhibited binding to disrupted membranes indicating that the NBMPR-sensitive nucleoside transporter probably has a thiol group located on the inner surface of the membrane. Exposure of rat erythrocyte membranes to UV light in the presence of [3H]NBMPR resulted in covalent radiolabeling of a membrane protein(s) (apparent Mr on SDS gel electropherograms of 62,000). Labeling of this protein was abolished in the presence of nitrobenzylthioguanosine. We conclude that nucleoside transport by rat erythrocytes occurs by two facilitated-diffusion systems which differ in their sensitivity to inhibition by both NBMPR andpCMBS.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Barlow, R.W. 1983. Biodata Handling with Microcomputers. Elsevier Biosoft, Cambridge

    Google Scholar 

  • Belt, J.A. 1983. Heterogeneity of nucleoside transport in mammalian cells. Two types of transport activity in L1210 and other cultured neoplastic cells.Mol. Pharmacol. 24:479–484

    PubMed  Google Scholar 

  • Belt, J.A., Noel, L.D. 1985. Nucleoside transport in Walker 256 rat carcinosarcoma and S49 mouse lymphoma cells. Differences in sensitivity to nitrobenzylthioinosine and sulfhydryl reagents.Biochem. J. 232:681–688

    PubMed  Google Scholar 

  • Beutler, E., West, C., Blume, K.G. 1976. The removal of leukocytes and platelets from whole blood.J. Lab. Clin. Med. 88:328–333

    PubMed  Google Scholar 

  • Bibi, O., Schwartz, J., Eilam, Y., Shohami, E., Cabantchik, Z.I. 1978. Nucleoside transport in mammalian cell membranes: IV. Organomercurials and organomercurial-mercaptonucleoside complexes as probes for nucleoside transport systems in hamster cells.J. Membrane Biol. 39:159–183

    Google Scholar 

  • Cass, C.E., Gaudette, L.A., Paterson, A.R.P. 1974. Mediated transport of nucleosides in human erythrocytes. Specific binding of the inhibitor nitrobenzylthioinosine to nucleoside transport sites in the erythrocyte membrane.Biochim. Biophys. Acta 345:1–10

    PubMed  Google Scholar 

  • Cass, C.E., Kolassa, N., Uehara, Y., Dahlig-Harley, E., Harley, E.R., Paterson, A.R.P. 1981. Absence of binding sites for the transport inhibitor nitrobenzylthioinosine on nucleoside transport-deficient mouse lymphoma cells.Biochim. Biophys. Acta 649:769–777

    PubMed  Google Scholar 

  • Dahlig-Harley, E., Eilam, Y., Paterson, A.R.P., Cass, C.E. 1981. Binding of nitrobenzylthioinosine to high-affinity sites on the nucleoside-transport mechanism of HeLa cells.Biochem. J. 200:295–305

    PubMed  Google Scholar 

  • Dodge, J.T., Mitchell, C., Hanahan, D. 1963. The preparation and chemical characteristics of haemoglobin-free ghosts of human erythrocytes.Arch. Biochem. Biophys. 100:119–130

    PubMed  Google Scholar 

  • Eilam, Y., Cabantchik, Z.I. 1977. Nucleoside transport in mammalian cell membranes: A specific inhibitory mechanism of high affinity probes.J. Cell. Physiol. 92:185–202

    Google Scholar 

  • Gati, W.P., Belt, J.A., Jakobs, E.S., Young, J.D., Jarvis, S.M., Paterson, A.R.P. 1986. Photoaffinity labelling of a nitrobenzylthioinosine-binding polypeptide from cultured Novikoff hepatoma cells.Biochem. J. (in press)

  • Hammond, J.R., Clanachan, A.S. 1984. [3H]Nitrobenzylthioinosine binding to the guinea pig CNS nucleoside transport system: A pharmacological characterization.J. Neurochem. 43:1582–1592

    PubMed  Google Scholar 

  • Heichal, O., Bibi, O., Katz, J., Cabantchik, Z.I. 1978. Nucleoside transport in mammalian cell membranes: III. Kinetic and chemical modification studies of cytozine-arabinoside and uridine transport in hamster cells in culture.J. Membrane Biol. 39:133–157

    Google Scholar 

  • Jarvis, S.M., Hammond, J.R., Paterson, A.R.P., Clanachan, A.S. 1982a. A study of uridine transport and nitrobenzylthioinosine binding by mammalian erythrocytes.Biochem. J. 208:83–88

    PubMed  Google Scholar 

  • Jarvis, S.M., Janmohamed, S.N., Young, J.D. 1983. Kinetics of nitrobenzylthioinosine binding to the human erythrocyte nucleoside transporter.Biochem. J. 216:661–667

    PubMed  Google Scholar 

  • Jarvis, S.M., Martin, B.W. 1986. Effects of temperature on the transport of nucleosides in guinea pig erythrocytes.Can. J. Physiol. Pharmacol. 64:193–198

    PubMed  Google Scholar 

  • Jarvis, S.M., McBride, D., Young, J.D. 1982b. Erythrocyte nucleoside transport: Asymmetrical binding of nitrobenzylthioinosine to nucleoside permeation sites.J. Physiol. (London) 324:31–46

    Google Scholar 

  • Jarvis, S.M., Young, J.D. 1980. Nucleoside transport in human and sheep erythrocytes. Evidence that nitrobenzylthioinosine binds specifically to functional nucleoside-transport sites.Biochem. J. 190:377–383

    PubMed  Google Scholar 

  • Jarvis, S.M., Young, J.D. 1982. Nucleoside translocation in sheep reticulocytes and fetal erythrocytes. A proposed model for the nucleoside transporter.J. Physiol. (London) 324:47–66

    Google Scholar 

  • Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature (London) 227:680–686

    Google Scholar 

  • Paterson, A.R.P., Jakobs, E.S., Harley, E.R., Cass, C.E., Robins, M.J. 1983. Inhibitors of nucleoside transport as probes and drugs.In: Development of Target-Oriented Anticancer Drugs. pp. 41–56. Y.C. Cheng, B. Goz, and M. Minkoff, editors. Raven, New York

    Google Scholar 

  • Plagemann, P.G.W., Wohlhueter, R.M. 1984a. Nucleoside transport in cultured mammalian cells. Multiple forms with different sensitivity to inhibition by nitrobenzylthioinosine or hypoxanthine.Biochim. Biophys. Acta 773:39–52

    PubMed  Google Scholar 

  • Plagemann, P.G.W., Wohlhueter, R.M. 1984b. Effect of sulfhydryl reagents on nucleoside transport in cultured mammalian cells.Arch. Biochem. Biophys. 233:489–500

    PubMed  Google Scholar 

  • Plagemann, P.G.W., Wohlhueter, R.M. 1985. Nitrobenzylthioinosine-sensitive and-resistant nucleoside transport in normal and transformed rat cells.Biochim. Biophys. Acta 816:387–395

    PubMed  Google Scholar 

  • Rothstein, A. 1970. Sulfhydryl groups in membrane structure and function.Curr. Top. Membr. Transp. 1:135–176

    Google Scholar 

  • Shi, M.M., Wu, J.S., Lee, C.M., Young, J.D. 1984. Nucleoside transport. Photoaffinity labelling of high-affinity nitrobenzylthioinosine binding sites in rat and guinea pig lung.Biochem. Biophys. Res. Commun. 118:594–600

    PubMed  Google Scholar 

  • Tse, C.M., Wu, J.S., Young, J.D. 1985. Evidence for the asymmetrical binding ofp-chloromercuriphenylsulphonate to the human erythrocyte nucleoside transporter.Biochim. Biophys. Acta 818:316–324

    PubMed  Google Scholar 

  • Verma, A., Marangos, P.J. 1985. Nitrobenzylthioinosine binding in brain: An interspecies study.Life Sci. 36:283–290

    PubMed  Google Scholar 

  • Williams, E.F., Barker, P.H., Clanachan, A.S. 1984. Nucleoside transport in heart: Species differences in nitrobenzylthioinosine binding, adenosine accumulation, and drug induced potentiation of adenosine action.Can. J. Physiol. Pharmacol. 62:31–37

    PubMed  Google Scholar 

  • Wu, J.S., Jarvis, S.M., Young, J.D. 1983a. Identification of the erythrocyte nucleoside transporter as a band 4.5 polypeptide. Photoaffinity labelling studies using nitrobenzylthioinosine.J. Biol. Chem. 258:13745–13751

    PubMed  Google Scholar 

  • Wu, J.S., Kwong, F.Y.P., Jarvis, S.M., Young, J.D. 1983b. The human erythrocyte nucleoside and glucose transporters are both band 4.5 membrane polypeptides.Biochem. J. 214:995–997

    PubMed  Google Scholar 

  • Wu, J.S.R., Young, J.D. 1984. Photoaffinity labelling of nucleoside-transport proteins in plasma membranes isolated from rat and guinea-pig liver.Biochem. J. 220:499–506

    PubMed  Google Scholar 

  • Young, J.D. 1978. Nucleoside transport in sheep erythrocytes: Genetically controlled transport variation and its influence on erythrocyte ATP concentrations.J. Physiol. (London) 277:325–339

    Google Scholar 

  • Young, J.D., Ellory, J.C. 1982. Flux measurements.In: Red Cell Membranes—A Methodological Approach. pp. 119–133. J.C. Ellory and J.D. Young, editors. Academic, London

    Google Scholar 

  • Young, J.D., Jarvis, S.M. 1983. Nucleoside transport in animal cells.Review. Biosci. Rep. 3:309–322

    Google Scholar 

  • Young, J.D., Jarvis, S.M., Belt, J.A., Gati, W.P., Paterson, A.R.P. 1984. Identification of the nucleoside transporter in cultured mouse lymphoma cells.J. Biol. Chem. 259:8363–8365

    PubMed  Google Scholar 

  • Young, J.D., Jarvis, S.M., Robins, M.J., Paterson, A.R.P. 1983. Photoaffinity labelling of human erythrocyte nucleoside transporter by N6-(p-azidobenzyl)-adenosine and nitrobenzylthioinosine.J. Biol. Chem. 258:2202–2208

    PubMed  Google Scholar 

Download references

Author information

Author notes

  1. Simon M. Jarvis

    Present address: Biological Laboratory, University of Kent, CT2 7NJ, Canterbury, Kent, U.K.

Authors and Affiliations

  1. Department of Physiology, University of Alberta, T6G 2H7, Edmonton, Alberta, Canada

    Simon M. Jarvis

  2. Department of Biochemistry, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong

    James D. Young

Authors
  1. Simon M. Jarvis
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. James D. Young
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Jarvis, S.M., Young, J.D. Nucleoside transport in rat erythrocytes: two components with differences in sensitivity to inhibition by nitrobenzylthioinosine andp-chloromercuriphenyl sulfonate. J. Membrain Biol. 93, 1–10 (1986). https://doi.org/10.1007/BF01871013

Download citation

  • Received:

  • Revised:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF01871013

Key Words

Search

Navigation