Advertisement

Journal of Bioenergetics and Biomembranes

, Volume 27, Issue 1, pp 31–36 | Cite as

ATP hydrolysis by multidrug-resistance protein from Chinese hamster ovary cells

  • Alan E. Senior
  • Marwan K. Al-Shawi
  • Ina L. Urbatsch
Article

Abstract

ATPase activity of multidrug-resistance protein (P-glycoprotein, Pgp) from Chinese hamster ovary cells was studied. Catalytic characteristics were established for Pgp both in its natural plasma membrane environment and in purified reconstituted protein. Generally the two preparations of Pgp behaved similarly, and demonstrated low affinity for MgATP, low nucleotide specificity, preference for Mg-nucleotide, and pH optimum near 7.5. A high-affinity binding site involved in catalysis was not apparent. Effective covalent inactivators were NBD-C1, NEM, 8-azido-ATP, and 2-azido-ATP. DCCD, FITC, and pyridoxal phosphate were only weakly inhibitory. Lipid composition was found to affect the degree of drug stimulation of ATPase in purified reconstituted Pgp, suggesting that the lipid environment affects coupling between drug-binding and catalytic sites, and that Pgp expressed in different tissues could show different functional characteristics.

Key words

MDR protein ATP hydrolysis catalytic sites 

Abbreviations

Nucleotide analogs

AMPPNP

adenyl-5′-yl-imidodiphosphate

TNP-ATP

2′,3′-O-(2,4,6)-trinitrophenyl-ATP

BzATP

3′-O-(4-benzoyl)benzoyl-ATP

ATPγS

adcnosine-5′-O-(3-thiotriphosphate)

Other compounds

DCCD

dicyclohexylcarbodiimide

DTT

dithiothreitol

FITC

fluorescein isothiocyanate

NEM

N-ethylmaleimide

NBD-Cl

7-chloro-4-nitrobenzo-2-oxa-1.3-diazole

TPA+

tetraphenylarsonium ion: TPP+, tetraphenylphosphonium ion

Pgp

P-glycoprotein

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Al-Shawi, M. K., and Senior, A. E. (1993).J. Biol. Chem. 268, 4197–4206.Google Scholar
  2. Al-Shawi, M. K., Urbatsch, I. L., and Senior, A. E. (1994).J. Biol. Chem. 269, 8986–8992.Google Scholar
  3. Ambudkar, S. V., Lelong, I. H., Zhang, J., Cardarelli, C. O., Gottesman, M. M., and Pastan, I. (1992).Proc. Natl. Acad. Sci. USA 89, 8472–8476.PubMedGoogle Scholar
  4. Azzaria, M., Schurr, E., and Gros, P. (1989).Mol. Cell. Biol. 9, 5289–5297.PubMedGoogle Scholar
  5. Bibi, E., Gros, P., and Kaback, H. R. (1993).Proc. Natl. Acad. Sci. USA 90, 9209–9213.PubMedGoogle Scholar
  6. Bradley, G., Juranka, P. F., and Ling, V. (1988).Biochim. Biophys. Acta 948, 87–128.PubMedGoogle Scholar
  7. Georges, E., Bradley, G., Garriepy, J., and Ling, V. (1990).Proc. Natl. Acad. Sci. USA 87, 152–156.PubMedGoogle Scholar
  8. Georges, E., Zhang, J.-T., and Ling, V. (1991).J. Cell Physiol. 148, 479–484.PubMedGoogle Scholar
  9. Gottesman, M. M., and Pastan, I. (1993).Annu. Rev. Biochem. 62, 385–427.PubMedGoogle Scholar
  10. Gros, P., Talbot, F., Tang-Wai, D., Bibi, E., and Kaback, H. R. (1992).Biochemistry 31, 1992–1998.PubMedGoogle Scholar
  11. Horio, M., Lovelace, E., Gottesman, M. M., and Pastan, I. (1991).Biochim. Biophys. Acta 1061, 106–110.PubMedGoogle Scholar
  12. Kartner, N., Evernden-Porelle, D., Bradley, G., and Ling, V. (1985).Nature (London) 316, 820–823.Google Scholar
  13. Loo, T. P., and Clarke, D. M. (1994).J. Biol. Chem. 269, 7750–7755.Google Scholar
  14. Rao, U. S., and Scarborough, G. A. (1994).Mol. Pharmacol. 45, 773–776.PubMedGoogle Scholar
  15. Riordan, J.R., and Ling, V. (1979).J. Biol. Chem. 254, 12701–12705.Google Scholar
  16. Ruetz, S., and Gros, P. (1994).J. Biol. Chem. 269, 12277–12284.Google Scholar
  17. Sarkadi, B., Price, E. M., Boucher, R. C., Germann, U. A., and Scarborough, G. A. (1992).J. Biol. Chem. 267, 4854–4858.Google Scholar
  18. Shapiro, A. B., and Ling, V. (1994).J. Biol. Chem. 269, 3745–3754.Google Scholar
  19. Sharom, F. J., Yu, X., and Doige, C. A. (1993).J. Biol. Chem. 268, 24197–24202.Google Scholar
  20. Tanford, C. (1983).Annu. Rev. Biochem. 52, 379–409.PubMedGoogle Scholar
  21. Urbatsch, I. L., and Senior, A. E. (1994).Arch. Biochem. Biophys. 316, 135–140.Google Scholar
  22. Urbatsch, I. L., Al-Shawi, M. K., and Senior, A. E. (1994).Biochemistry 33, 7069–7076.PubMedGoogle Scholar
  23. Walker, J. E., Saraste, M., Runswick, M. J., and Gay, N. J. (1982).EMBO J. 1, 945–951.PubMedGoogle Scholar

Copyright information

© Plenum Publishing Corporation 1995

Authors and Affiliations

  • Alan E. Senior
    • 1
  • Marwan K. Al-Shawi
    • 1
  • Ina L. Urbatsch
    • 1
  1. 1.Department of BiochemistryUniversity of Rochester Medical CenterRochester

Personalised recommendations