Skip to main content
SpringerLink
Log in

Isolation and reconstitution of furosemide-binding proteins from Ehrlich ascites tumor cells

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

Summary

Furosemide-binding proteins were isolated from cholate-solubilized membranes of Ehrlich ascites tumor cells by affinity chromatography, using furosemide as ligand. Solubilized proteins retarded by the affinity material were eluted by furosemide. In reducing and denaturing gels, the major proteins eluted by furosemide were 100 and 45 kDa. In nonreducing, nondenaturing gels, homodimers of both polypeptides were found, whereas no oligomeric proteins containing both polypeptides were seen. It is concluded that the furosemide gel binds two distinct dimeric proteins. The isolated proteins were reconstituted into phospholipid vesicles and the K+ transport activity of these vesicles was assayed by measurement of86Rb+ uptake against a large opposing K+ gradient. The reconstituted system was found to contain a K+ transporting protein, which is sensitive to Ba2+ like the K+ channel previously demonstrated to be activated in intact cells after cell swelling.

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

  • Allen, R.C. 1983. Sulfonamide diuretics.In: Diuretics. E.J. Cragoe, editor. pp. 119–200. John Wiley, New York

    Google Scholar 

  • Aull, F. 1982. Specific drug sensitive transport pathways for chloride and potassium ions in steady-state Ehrlich mouse ascites tumor cells.Biochim. Biophys. Acta 688:740–746

    PubMed  Google Scholar 

  • Aull, F., Nachbar, M.S., Oppenheim, J.D. 1977. Chloride self exchange in Ehrlich ascites cells. Inhibition by furosemide and 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid.Biochim. Biophys. Acta 471:341–347

    PubMed  Google Scholar 

  • Brazy, P.C., Gunn, R.B. 1976. Purosemide inhibition of chloride transport in human red blood cells.J. Gen. Physiol. 68:583–599

    Article  PubMed  Google Scholar 

  • Cherksey, B.D., Hoffmann, E.K., Jessen, F. Zeuthen, T. 1987. The K+ and Cl− channel protein from Ehrlich ascites tumour cells under reducing and oxidizing conditions.J. Physiol. (London) 390:223P

    Google Scholar 

  • Cherksey, B.D., Zeuthen, T. 1987. A membrane protein with a K+ and Cl− channel.Acta Physiol. Scand. 129:137–138

    PubMed  Google Scholar 

  • Cherksey, B.D., Zeuthen, T. 1988. H-bumetanide binding to the purified putative co-transporter protein.Acta Physiol. Scand. 133:267–268

    PubMed  Google Scholar 

  • Chipperfield, A.R. 1980. An effect of chloride on (Na+K) co-transport in human red blood cells.Nature (London) 286:281–282

    Article  Google Scholar 

  • Chipperfield, A.R. 1986. The (Na+-K+-Cl−) co-transport system.Clin. Sci. 71:465–476

    PubMed  Google Scholar 

  • Dunham, P.B., Ellory, J.C. 1981. Passive potassium transport in low potassium sheep red cells: Dependence upon cell volume and chloride.J. Physiol. (London) 318:511–530

    Google Scholar 

  • Dunham, P.B., Stewart, G.W., Ellory, J.C. 1980. Chloride-activated passive potassium transport in human erythrocytes.Proc. Natl. Acad. Sci. USA 77:1711–1715

    PubMed  Google Scholar 

  • Ellory, J.C., Dunham, P.B., Logue, P.J., Stewart, G.W. 1982. Anion-dependent cation transport in erythrocytes.Phil. Trans. R. Soc. London (B) 299:483–495

    Google Scholar 

  • Ellory, J.C., Hall, A.C., Stewart, G.W. 1985. Volume-sensitive cation fluxes in mammalian red cells.Mol. Physiol. 8:235–246

    Google Scholar 

  • Feit, P.W., Hoffmann, E.K., Schiødt, M., Kristensen, P., Jessen, F., Dunham, P.B. 1988. Purification of proteins of the Na/Cl cotransporter from membranes of Ehrlich ascites cells using a bumetanide-sepharose affinity column.J. Membrane Biol. 103:135–147

    Google Scholar 

  • Frizzell, R.A., Field, M., Schultz, S.G. 1979. Sodium-coupled chloride transport by epithelial tissues.Am. J. Physiol. 236:F1-F8

    Google Scholar 

  • Garty, H., Rudy, B., Karlish, S.J.D. 1983. A simple and sensitive procedure for measuring isotope fluxes through ion-specific channels in heterogenous populations of membrane vesicles.J. Biol. Chem. 258:13094–13099

    PubMed  Google Scholar 

  • Geck, P., Heinz, E. 1986. The Na-K-2Cl cotransport system.J. Membrane Biol. 91:91–105

    Google Scholar 

  • Geck, P., Pietrzyk, C., Burckhardt, B.-C., Pfeiffer, B., Heinz, E. 1980. Electrically silent contransport of Na+, K+ and Cl− in Ehrlich cells.Biochim. Biophys. Acta 600:432–447

    PubMed  Google Scholar 

  • Görg, A., Postel, W., Weser, J., Schiwaro, H.W.; Boesken, W.H. 1985. Horizontal SDS electrophoresis in ultrathin poregradient gels for the analysis of urinary proteins.Science Tools 32:5–9

    Google Scholar 

  • Greger, R. 1985. Ion transport mechanisms in the thick ascending limb of Henle's loop of mammalian nephron.Physiol. Rev. 65:760–797

    PubMed  Google Scholar 

  • Haas, M., Forbush, B., III. 1987. Na, K, Cl-cotransport system: Characterization by bumetanide binding and photolabeling.Kidney Int. 32 (Suppl. 23):S134-S140

    Google Scholar 

  • Haas, M., McManus, T.J. 1985. Effect of norepinephrine on swelling-induced potassium transport in duck red cells. Evidence against a volume-regulatory decrease under physiological conditions.J. Gen. Physiol. 85:649–667

    PubMed  Google Scholar 

  • Helenius, A., Simons, K. 1972. The binding of detergents to lipophilic and hydrophilic proteins.J. Biol. Chem. 247:3656–3661

    PubMed  Google Scholar 

  • Hoffmann, E.K., Lambert, I.H., Simonsen, L.O. 1986a. Separate, Ca2+-activated K+ and Cl− transport pathways in Ehrlich ascites tumor cells.J. Membrane Biol. 91:227–244

    Article  Google Scholar 

  • Hoffmann, E.K., Schiødt, M., Dunham, P.B. 1986b. The number of chloride-cation cotransport sites on Ehrlich ascites cells measured with [3H]bumetanide.Am. J. Physiol. 250:C688-C693

    Google Scholar 

  • Hoffmann, E.K., Simonsen, L.O. 1989. Membrane mechanisms in volume and pH regulation in vertebrate cells.Physiol. Rev. (in press)

  • Hoffmann, E.K., Simonsen, L.O., Lambert, I.H. 1984. Volume-induced increase of K+ and Cl− permeabilities in Ehrlich ascites tumor cells. Role of internal Ca2+.J. Membrane Biol. 78:211–222

    Google Scholar 

  • Hoffmann, E.K., Simonsen, L.O., Sjøhlm, C. 1979. Membrane potential, chloride exchange, and chloride conductance in Ehrlich mouse ascites tumour cells.J. Physiol. (London) 296:61–84

    Google Scholar 

  • Hoffmann, E.K., Sjøhlm, C., Simonsen, L.O. 1983. Na+, Cl− co-transport in Ehrlich ascites tumor cells activated during volume regulation (regulatory volume increase).J. Membrane Biol. 76:269–280

    Article  Google Scholar 

  • Jessen, F., Sjøholm, C., Hoffmann, E.K. 1986. Identification of the anion exchange protein of Ehrlich cells: A kinetic analysis of the inhibitory effects of 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS) and labeling of membrane proteins with3H-DIDS.J. Membrane Biol. 92:195–205

    Google Scholar 

  • Kagawa, Y. 1972. Reconstitution of oxidative phosphorylation.Biochim. Biophys. Acta 265:297–338

    PubMed  Google Scholar 

  • Kinne, R.K.H., Kinne-Saffran, E., Schoelermann, B., Schuetz, H., Doell, G. 1987. Functional molecular size of rabbit kidney Na, K, Cl cotransporter.Kidney Int. 31:171

    Google Scholar 

  • Klaerke, D.A., Petersen, J., Jørgensen, P.L. 1987. Purification of Ca2+-activated K+ channel protein on calmodulin affinity columns after detergent solubilization of luminal membranes from outer renal medulla.FEBS Lett 216:211–216

    PubMed  Google Scholar 

  • Kramhøft, B., Lambert, I.H., Hoffmann, E.K., Jørgensen, F. 1986. Activation of Cl-dependent K transport in Ehrlich ascites tumor cells.Am. J. Physiol. 251:C369-C379

    PubMed  Google Scholar 

  • Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature (London) 227:680–685

    Google Scholar 

  • Lambert, I.H., Simonsen, L.O., Hoffmann, E.K. 1984. Volume regulation in Ehrlich ascites tumour cells: pH sensitivity of the regulatory volume decrease, and role of the Ca2+-dependent K+ channel.Acta Physiol. Scand. 120:46A

    Google Scholar 

  • Lauf, P.K. 1985a. On the relationship between volume- and thiol-stimulated K+ Cl− fluxes in red cell membranes.Mol. Physiol. 8:215–234

    Google Scholar 

  • Lauf, P.K. 1985b. K+:Cl− cotransport: Sulfhydryls, divalent cations, and the mechanism of volume activation in a red cell.J. Membrane Biol. 88:1–13

    Google Scholar 

  • Lauf, P.K., McManus, T.J., Haas, M., Forbush, B., III., Duhm, J., Flatman, P.W., Saier, M.H., Jr., Russel, J.M. 1987. Physiology and biophysics of chloride and cation cotransport across cell membranes.Fed. Proc. 46:2377–2394

    PubMed  Google Scholar 

  • Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. 1951. Protein measurement with the Folin phenol reagent.J. Biol. Chem. 193:265–275

    PubMed  Google Scholar 

  • McManus, T.J., Haas, M., Starke, L.C., Lytle, C.Y. 1985. The duck red cell model of volume-sensitive chloride-dependent cation transport:In: Membrane Transport Driven by Ion Gradients. G. Semenza and R. Kinne, editors.Ann. NY Acad. Sci. 456:183–186

    PubMed  Google Scholar 

  • McManus, T.J., Schmidt, W.F., III. 1978. Ion and co-ion transport in avian red cells.In: Membrane Transport Processes. J.F. Hoffmann, editor. Vol. 1, pp. 79–106. Raven, New York

    Google Scholar 

  • O'Grady, S.M., Palfrey, H.C., Field, M. 1987. Characteristics and functions of Na-K-Cl cotransport in epithelial tissues.Am. J. Physiol. 253:C177-C192

    Google Scholar 

  • Peterson, G.L. 1977. A simplification of the protein assay method of Lowry et al. which is more generally applicable.Anal. Biochem. 83:346–356

    Article  PubMed  Google Scholar 

  • Rodbard, D., Kapadia, G., Chramback, A. 1971. Pore gradient electrophoresis.Anal. Biochem. 40:135–157

    PubMed  Google Scholar 

  • Schubert, D., Boss, K., Dorst, H.-J., Flossdorf, J., Pappert, G. 1983. The nature of the stable noncovalent dimers of band 3 protein from erythrocyte membranes in solutions of Triton X-100.FEBS Lett. 163:81–84

    PubMed  Google Scholar 

  • Steck, T.L. 1972. Cross-linking the major proteins of the isolated erythrocyte membrane.J. Mol. Biol. 66:295–305

    PubMed  Google Scholar 

  • Tzagoloff, A., Penefsky, H.S. 1971. Extraction and purification of lipoprotein complexes from membranes.In: Methods in Enzymology, W.B. Jakoby, editor. Vol. 22, pp, 219–230. Academic, New York and London

    Google Scholar 

  • Warnock, D.G., Greger, R., Dunham, P.B., Benjamin, M.A., Frizzell, R.A., Field, M., Spring, K.R., Ives, H.E., Aronson, P.S., Seifter, J. 1984. Ion transport processes in apical membranes of epithelia.Fed. Proc. 43:2473–2487

    PubMed  Google Scholar 

  • Zeuthen, T., Andersen, P.M., Eskesen, K.E., Cherksey, B.D. 1988. Characterization of a purified co-transporting protein.Comp. Biochem. Physiol. 90:687–691

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Institute of Biological Chemistry A, August Krogh Institute, University of Copenhagen, DK-2100, Copenhagen Ø, Denmark

    Flemming Jessen & Else K. Hoffmann

  2. Department of Physiology and Biophysics, New York University School of Medicine, New York, New York

    Bruce D. Cherksey

  3. Institute of Medical Physiology, University of Copenhagen, DK-2200, Copenhagen N, Denmark

    Thomas Zeuthen

Authors
  1. Flemming Jessen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Bruce D. Cherksey
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Thomas Zeuthen
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Else K. Hoffmann
    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

Jessen, F., Cherksey, B.D., Zeuthen, T. et al. Isolation and reconstitution of furosemide-binding proteins from Ehrlich ascites tumor cells. J. Membrain Biol. 108, 139–151 (1989). https://doi.org/10.1007/BF01871025

Download citation

  • Received:

  • Revised:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF01871025

Key Words

Search

Navigation