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The Journal of Membrane Biology

, Volume 90, Issue 2, pp 123–135 | Cite as

A new method for the reconstitution of the anion transport system of the human erythrocyte membrane

  • Uwe Scheuring
  • Klaus Kollewe
  • Winfried Haase
  • Dieter Schubert
Articles

Summary

The anion transport protein of the human erythrocyte membrane, band 3, was solubilized and purified in solutions of the non-ionic detergent Triton X-100. It was incorporated into spherical lipid bilayers by the following procedure: (1) Dry phosphatidylcholine was suspended in the protein solution. Octylglucopyranoside was added until the milky suspension became clear. (2) The sample was dialyzed overnight against detergentfree buffer. (3) Residual Triton X-100 was removed from the opalescent vesicle suspension by sucrose density gradient centrifugation and subsequent dialysis. Sulfate efflux from the vesicles was studied, under exchange conditions, using a filtration method. Three vesicle subpopulations could be distinguished by analyzing the time course of the efflux. One was nearly impermeable to sulfate, and efflux from another was due to leaks. The largest subpopulation, however, showed transport characteristics very similar to those of the anion transport system of the intact erythrocyte membrane: transport numbers (at 30°C) close to 20 sulfate molecules per band 3 and min, an activation energy of approx. 140 kJ/mol, a pH maximum at pH 6.2, saturation of the sulfate flux at sulfate concentrations around 100mm, inhibition of the flux by H2DIDS and flufenamate (approx.Kl-values at 30°C: 0.1 and 0.7 μm, respectively), and “right-side-out” orientation of the transport protein (as judged from the inhibition of sulfate efflux by up to 98% by externally added H2DIDS). Thus, the system represents, for the first time, a reconstitution of all the major properties of the sulfate transport across the erythrocyte membrane.

Key Words

anion transport system human erythrocyte membrane band 3 protein reconstitution right-side-out vesicles 

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Copyright information

© Springer-Verlag New York Inc. 1986

Authors and Affiliations

  • Uwe Scheuring
    • 1
  • Klaus Kollewe
    • 1
  • Winfried Haase
    • 1
  • Dieter Schubert
    • 1
  1. 1.Max-Planck-Institut für BiophysikFrankfurt am Main 71West Germany

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