The Journal of Membrane Biology

, Volume 92, Issue 3, pp 195–205

Identification of the anion exchange protein of ehrlich cells: A kinetic analysis of the inhibitory effects of 4,4′-diisothiocyano-2,2′-stilbene-disulfonic acid (DIDS) and labeling of membrane proteins with3H-DIDS

  • Flemming Jessen
  • Carsten Sjøholm
  • Else K. Hoffmann
Articles

DOI: 10.1007/BF01869388

Cite this article as:
Jessen, F., Sjøholm, C. & Hoffmann, E.K. J. Membrain Biol. (1986) 92: 195. doi:10.1007/BF01869388

Summary

In Ehrlich ascites tumor cells 4,4′-diisothiocyano-2,2′-stillbene-disulfonic acid (DIDS) inhibits the chloride exchange both reversibly and irreversibly. The reversible inhibition is practically instantaneous and of a competitive nature withK1 about 2 μm at zero chloride concentration. This is succeeded by a slow irreversible binding of DIDS to the transporter, with a chloride dependence suggesting binding to the same site as for reversible DIDS binding/inhibition. To identify the membrane protein involved in anion exchange, cells were labeled with3H-DIDS. Incubation of cells for 10 min with 25 μm DIDS at pH 8.2 leads to more than 95% inhibition of the DIDS-sensitive chloride exchange flux when the chloride concentration is low (15mm). This condition was used for the3H-DIDS-labeling experiments. After incubation the cells were disrupted, the membranes isolated and solubilized, and the proteins separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The distribution of the3H-activity in the gel showed only one major peak, which could be related to protein with a mol wt of about 30,000 Daltons. The number of transport sites was estimated at about 400,000 per cell, and from the DIDS-sensitive chloride flux under steady-state conditions we calculate a turnover number of 340 ions per sec per site.

Key Words

anion exchangeDIDSEhrlich ascites tumor cellschloride fluxes

Copyright information

© Springer-Verlag New York Inc. 1986

Authors and Affiliations

  • Flemming Jessen
    • 1
  • Carsten Sjøholm
    • 2
  • Else K. Hoffmann
    • 1
  1. 1.Institute of Biological Chemistry A, August Krogh InstituteUniversity of CopenhagenCopenhagen ØDenmark
  2. 2.Novo Industri A/SBagsvaerdDenmark