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P-Type ATPases pp 127–156Cite as

Assaying P-Type ATPases Reconstituted in Liposomes

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1377))

Abstract

Reconstitution of P-type ATPases in unilamellar liposomes is a useful technique to study functional properties of these active ion transporters. Experiments with such liposomes provide an easy access to substrate-binding affinities of the ion pumps as well as to the lipid and temperature dependence of the pump current. Here, we describe two reconstitution methods by dialysis and the use of potential-sensitive fluorescence dyes to study transport properties of two P-type ATPases, the Na,K-ATPase from rabbit kidney and the K+-transporting KdpFABC complex from E. coli. Several techniques are introduced how the measured fluorescence signals may be analyzed to gain information on properties of the ion pumps.

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References

  1. Goldin SM, Tong SW (1974) Reconstitution of active transport catalyzed by the purified sodium and potassium ion-stimulated adenosine triphosphatase from canine renal medulla. J Biol Chem 249:5907–5915

    PubMed  CAS  Google Scholar 

  2. Anner BM, Lane LK, Schwartz A, Pitts BJR (1977) A reconstituted Na++K+ pump in liposomes containing purified (Na++K+)-ATPase from kidney medulla. Biochim Biophys Acta 467:340–345

    Article  PubMed  CAS  Google Scholar 

  3. Karlish SJD, Pick U (1981) Sidedness of the effects of sodium and potassium ions on the conformational state of the sodium-potassium pump. J Physiol 312:505–529

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  4. Brotherus JR, Jacobsen L, Jørgensen PL (1983) Soluble and enzymatically stable (Na+ + K+)-ATPase from mammalian kidney consisting predominantly of protomer αβ-units. Preparation, assay and reconstitution of active Na+, K+ transport. Biochim Biophys Acta 731:290–303

    Article  PubMed  CAS  Google Scholar 

  5. Cornelius F, Skou JC (1984) Reconstitution of (Na++K+)-ATPase into phospholipid vesicles with full recovery of its specific activity. Biochim Biophys Acta 772:357–373

    Article  PubMed  CAS  Google Scholar 

  6. Forbush B III (1984) An apparatus for rapid kinetic analysis of isotopic efflux from membrane vesicles and of ligand dissociation from membrane proteins. Anal Biochem 140:495–505

    Article  PubMed  CAS  Google Scholar 

  7. Apell H-J, Marcus MM, Anner BM, Oetliker H, Läuger P (1985) Optical study of active ion transport in lipid vesicles containing reconstituted Na K-ATPase. J Membr Biol 85:49–63

    Article  PubMed  CAS  Google Scholar 

  8. Fürst P, Solioz M (1986) The vanadate-sensitive ATPase of Streptococcus faecalis pumps potassium in a reconstituted system. J Biol Chem 261:4302–4308

    Google Scholar 

  9. Fendler K, Drose S, Altendorf K, Bamberg E (1996) Electrogenic K+ transport by the Kdp-ATPase of Escherichia coli. Biochemistry 35:8009–8017

    Article  PubMed  CAS  Google Scholar 

  10. Anner BM, Moosmayer M (1981) Preparation of Na, K-ATPase-containing liposomes with predictable transport properties by a procedure relating the Na, K-transport capacity to the ATPase activity. J Biochem Biophys Meth 5:299–306

    Article  Google Scholar 

  11. Forbush B III (1984) Na+ movement in a single turnover of the Na pump. Proc Natl Acad Sci U S A 81:5310–5314

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  12. Anner BM, Marcus MM, Moosmayer M (1984) Reconstitution of Na, K-ATPase. In: Azzi A, Brodbeck U, Zahler P (eds) Enzymes, receptors and carriers of biological membranes – a laboratory manual. Springer, Heidelberg, pp 81–96

    Chapter  Google Scholar 

  13. Goldshlegger R, Karlish SJ, Rephaeli A, Stein WD (1987) The effect of membrane potential on the mammalian sodium-potassium pump reconstituted into phospholipid vesicles. J Physiol 387:331–355

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  14. Clarke RJ, Apell H-J (1989) A stopped-flow kinetic study of the interaction of potential-sensitive oxonol dyes with lipid vesicles. Biophys Chem 34:225–237

    Article  PubMed  CAS  Google Scholar 

  15. Damnjanovic B, Weber A, Potschies M, Greie JC, Apell H-J (2013) Mechanistic analysis of the pump cycle of the KdpFABC P-type ATPase. Biochemistry 52:5563–5576

    Google Scholar 

  16. Bashford CL, Chance B, Smith JC, Yoshida T (1979) The behavior of oxonol dyes in phospholipid dispersions. Biophys J 25:63–85

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  17. Apell H-J, Bersch B (1987) Oxonol VI as an optical indicator for membrane potentials in lipid vesicles. Biochim Biophys Acta 903:480–494

    Article  PubMed  CAS  Google Scholar 

  18. Clarke RJ, Apell H-J, Läuger P (1989) Pump current and Na+/K+ coupling ratio of Na+/K+-ATPase in reconstituted lipid vesicles. Biochim Biophys Acta 981:326–336

    Article  PubMed  CAS  Google Scholar 

  19. Jørgensen PL (1974) Isolation of (Na++K+)-ATPase. Meth Enzymol 32:277–290

    Article  PubMed  Google Scholar 

  20. Jørgensen PL (1982) Mechanism of the Na+, K+ pump. Protein structure and conformations of the pure (Na++K+)-ATPase. Biochim Biophys Acta 694:27–68

    Google Scholar 

  21. Schwartz AK, Nagano M, Nakao M, Lindenmayer GE, Allen JC (1971) The sodium- and potassium-activated adenosinetriphosphatase system. Meth Pharmacol 1:361–388

    Article  CAS  Google Scholar 

  22. Vagin O, Denevich S, Munson K, Sachs G (2002) SCH28080, a K + -competitive inhibitor of the gastric H, K-ATPase, binds near the M5–6 luminal loop, preventing K+ access to the ion binding domain. Biochemistry 41:12755–12762

    Google Scholar 

  23. Lowry OH, Rosebrough AL, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275

    PubMed  CAS  Google Scholar 

  24. Apell H-J, Marcus MM (1985) Effects of vesicle inhomogeneity on the interpretation of flux data obtained with reconstituted Na, K-ATPase. In: Glynn J, Ellory C (eds) The sodium pump. The Company of Biologists Ltd., Cambridge, pp 475–480

    Google Scholar 

  25. Apell H-J, Läuger P (1986) Quantitative analysis of pump-mediated fluxes in reconstituted lipid vesicles. Biochim Biophys Acta 861:302–310

    Article  CAS  Google Scholar 

  26. Apell H-J, Häring V, Roudna M (1990) Na, K-ATPase in artificial lipid vesicles. Comparison of Na, K and Na-only pumping mode. Biochim Biophys Acta 1023:81–90

    Article  PubMed  CAS  Google Scholar 

  27. Hoffman JF, Laris PC (1974) Determination of membrane potentials in human and Amphiuma red blood cells by means of fluorescent probe. J Physiol 239:519–552

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  28. Damnjanovic B, Apell H-J (2014) KdpFABC reconstituted in E. coli lipid vesicles: substrate dependence of the transport rate. Biochemistry 53(35):5674–5682

    Google Scholar 

  29. Cirri E, Kirchner C, Becker S, Katz A, Karlish SJ, Apell H-J (2013) Surface charges of the membrane crucially affect regulation of Na, K-ATPase by phospholemman (FXYD1). J Membr Biol 246:967–979

    Google Scholar 

  30. Markwell MA, Haas SM, Bieber LL, Tolbert NE (1978) A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal Biochem 87:206–210

    Article  PubMed  CAS  Google Scholar 

  31. Marcus MM, Apell H-J, Roudna M, Schwendener RA, Weder HG, Läuger P (1986) (Na++ K+)-ATPase in artificial lipid vesicles: influence of lipid structure on pumping rate. Biochim Biophys Acta 854:270–278

    Article  PubMed  CAS  Google Scholar 

  32. Waggoner AS (1979) Dye indicators of membrane potential. Annu Rev Biophys Bioeng 8:47–68

    Article  PubMed  CAS  Google Scholar 

  33. Bashford CL (1981) The measurement of membrane potential using optical indicators. Biosci Rep 1:183–196

    Article  PubMed  CAS  Google Scholar 

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Acknowledgement

This work was supported by the Konstanz Research School Chemical Biology, University of Konstanz, Germany, and the University of Konstanz (AFF 4/68).

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Authors and Affiliations

  1. Department of Biology, University of Konstanz, 635, Universitätsstr. 10, Konstanz, 78464, Germany

    Hans-Jürgen Apell & Bojana Damnjanovic

Authors
  1. Hans-Jürgen Apell
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  2. Bojana Damnjanovic
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Correspondence to Hans-Jürgen Apell .

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Editors and Affiliations

  1. Department of Biochemistry, Biocenter, University of Oxford, Oxford, Oxfordshire, United Kingdom

    Maike Bublitz

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Apell, HJ., Damnjanovic, B. (2016). Assaying P-Type ATPases Reconstituted in Liposomes. In: Bublitz, M. (eds) P-Type ATPases. Methods in Molecular Biology, vol 1377. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3179-8_14

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  • DOI: https://doi.org/10.1007/978-1-4939-3179-8_14

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3178-1

  • Online ISBN: 978-1-4939-3179-8

  • eBook Packages: Springer Protocols

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