Skip to main content
SpringerLink
Log in

Reconstitution of hepatic uricase in planar lipid bilayer reveals a functional organic anion channel

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

Abstract

Rat renal proximal tubule cell membranes have been reported to contain uricase-like proteins that function as electrogenic urate transporters. Although uricase, per se, has only been detected within peroxisomes in rat liver (where it functions as an oxidative enzyme) this protein has been shown to function as a urate transport protein when inserted into liposomes. Since both the uricase-like renal protein and hepatic uricase can transport urate, reconstitution studies were performed to further characterize the mechanism by which uricase may function as a transport protein. Ion channel activity was evaluated in planar lipid bilayers before and after fusion of uricase-containing proteoliposomes. In the presence of symmetrical solutions of urate and KCl, but absence of uricase, no current was generated when the voltage was ramped between ±100 mV. Following fusion of uricase with the bilayer, single channel activity was evident: the reconstituted channel rectified with a mean slope conductance of 8 pS, displayed voltage sensitivity, and demonstrated a marked selectivity for urate relative to K+ and Cl−. The channel was more selective to oxonate, an inhibitor of both enzymatic uricase activity and urate transport, than urate and it was equally selective to urate and pyrazinoate, an inhibitor of urate transport. With time, pyrazinoate blocked both its own movement and the movement of urate through the channel. Channel activity was also blocked by the IgG fraction of a polyclonal antibody to affinity purified pig liver uricase. These studies demonstrate that a highly selective, voltage dependent organic anion channel is formed when a purified preparation of uricase is reconstituted in lipid bilayers.

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

  1. Abramson, R.G., King, V.F., Reif, M.C., Leal-Pinto, E., Baruch, S.B. 1982. Urate uptake in membrane vesicles of rat renal cortex: effect of copper. Am. J. Physiol. 242:F158-F170

    Google Scholar 

  2. Abramson, R.G., Lipkowitz, M.S. 1985. Carrier-mediated concentrative urate transport in renal membrane vesicles. Am. J. Physiol. 17:F574-F584

    Google Scholar 

  3. Abramson, R.G., Lipkowitz, M.S. 1990. Evolution of the uric acid transport mechanisms in vertebrate kidney. In: Basic Principles in Transport. R.K.H. Kinne, editor, pp. 115–153. Karger, Basal, Switzerland

    Google Scholar 

  4. Alton, E.W., Manning, S.D., Schlatter, P.J., Geddes, D.M., Williams, A.J. 1991. Characterization of a Ca(2+)-dependent anion channel from sheep tracheal epithelium incorporated into planar bilayers. J. Physiol. Lond. 443:137–159

    Google Scholar 

  5. Banderali, U., Roy, G. 1992. Anion channels for amino acids in MDCK cells. Am. J. Physiol. 263:C1200-C1207

    Google Scholar 

  6. Blomstedt, J.W., Aronson, P.S. 1980. pH gradient-stimulated transport of urate and p-aminohippurate in dog renal microvillus membrane vesicles. J. Clin. Invest. 65:931–934

    Google Scholar 

  7. Bureau, M.H., Khestchatisky, M., Heeren, M.A., Zambrowicz, E.B., Kim, H., Grisar, T.M., Colombini, M., Tobin, A.J., Olsen, R.W. 1992. Isolation and cloning of a voltage-dependent anion channel-like Mr 36,000 polypeptide from mammalian brain. J. Biol. Chem. 267:8679–8684

    Google Scholar 

  8. Chou, P.Y., Fasman, G.D. 1978. Prediction of the secondary structure of proteins from their amino acid sequences. Adv. Enzym. 47:45–148

    Google Scholar 

  9. De Duve, C., Baudhuin, P. 1966. Peroxisomes (microbodies and related particles). Physiol. Rev. 46:323–357

    Google Scholar 

  10. Duszyk, M., Liu, D., French, A.S., Man, S.F. 1993. Halide permeation through three types of epithelial anion channels after reconstitution into giant liposomes. Eur. Biophys. J. 22:5–11.

    Google Scholar 

  11. Fridovich, I. 1965. The competitive inhibition of uricase by oxonate and by related derivatives of s-triazines. J. Biol. Chem. 240:2491–2494

    Google Scholar 

  12. Goldman, D.E. 1943. Potential, impedance and rectification in membranes. J. Gen. Physiol. 27:37–60.

    Google Scholar 

  13. Guggino, S.E., Martin, G.J., Aronson, P.S. 1983. Specificity and modes of the anion exchanger in dog renal microvillus membranes. Am. J. Physiol. 244:F612-F621

    Google Scholar 

  14. Hayman, K.A., Spurway, T.D., Ashley, R.H. 1993. Single anion channels reconstituted from cardiac mitoplasts. J. Membrane Biol. 136:181–190

    Google Scholar 

  15. Hedrich, R., Jeromin, A. 1992. A new scheme of symbiosis: ligandand voltage-gated anion channels in plants and animals. Philos. Trans. R. Soc. Lond. Biol. 338:31–38

    Google Scholar 

  16. Hedrich, R., Marten, I. 1993. Malate-induced feedback regulation of plasma membrane anion channels could provide a CO2 sensor to guard cells. EMBO J. 12:897–901

    Google Scholar 

  17. Hille, B. 1984. Classical biophysics of the squid giant axon. In: Ionic Channels of Excitable Membranes, pp. 23–57. Sinauer, Sunderland, MA

    Google Scholar 

  18. Hruban, Z., Rechcigl, Jr., M. 1969. Microbodies of various animal species. In: Microbodies and Related Particles. G.H. Bourne and J.F. Danielli, editors, pp. 20–39. Academic Press, New York

    Google Scholar 

  19. Jackson, P.S., Strange, K. 1993. Volume-sensitive anion channels mediate swelling-activated inositol and taurine efflux. Am. J. Physiol. 265:C1489-C1500

    Google Scholar 

  20. Kahn, A.M., Aronson, P.S. 1983. Urate transport via anion exchange in dog renal microvillus membrane vesicles. Am. J. Physiol. 244:F56-F63

    Google Scholar 

  21. Kahn, A.M., Branham, S., Weinman, E.J. 1983. Mechanism of urate and p-aminohippurate transport in rat renal microvillus membrane vesicles. Am. J. Physiol. 245:F151-F158

    Google Scholar 

  22. Kahn, A.M., Shelat, H., Weinman, E.J. 1985. Urate and p-aminohippurate transport in rat renal basolateral vesicles. Am. J. Physiol. 249:F684-F661

    Google Scholar 

  23. Knorr, B.A., Beck, J.C., Abramson, R.G. 1994. Classical and channel-like urate transporters in rabbit renal brush border membranes. Kid. Intern. 45:727–736

    Google Scholar 

  24. Knorr, B.A., Lipkowitz, M.S., Potter, B.J., Masur, S.K., Abramson, R.G. 1994. Isolation and immunolocalization of a rat renal cortical membrane urate transporter. J. Biol. Chem. 269:6759–6764

    Google Scholar 

  25. Kyte, J., Doolittle, R.F. 1982. A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157:105–132

    Google Scholar 

  26. Mahler, H.R., Baum, H.M., Hubscher, G. 1956. Enzymatic oxidation of urate. Science 124:705–708

    Google Scholar 

  27. McGill, J.M., Basavappa, S., Fitz, J.G. 1992. Characterization of high-conductance anion channels in rat bile duct epithelial cells. Am. J. Physiol. 262:G703-G710

    Google Scholar 

  28. McGill, J.M., Gettys, T.W., Basavappa, S., Fitz, J.G. 1993. GTPbinding proteins regulate high conductance anion channels in rat bile duct epithelial cells. J. Membrane Biol. 133:253–261

    Google Scholar 

  29. Moczydlowski, E., Latorre, R. 1983. Gating kinetics of Ca2+ activated K+ channels from rat muscle incorporated into planar lipid bilayers. J. Gen. Physiol. 82:511–542

    Google Scholar 

  30. Morales, E., de la Torre, L., Moy, G.W., Vacquier, V.D., Darszon, A. 1993. Anion channels in the sea urchin sperm plasma membrane. Mol. Reprod. Dev. 36:174–182

    Google Scholar 

  31. Oblatt-Montal, M., Reddy, G.L., Iwamoto, T., Tomich, J.M., Montai, M. 1994. Identification of an ion channel-forming motif in the primary structure of CFTR, the cystic fibrosis chloride channel. Proc. Natl. Acad. Sci. USA 91:1495–1499

    Google Scholar 

  32. Pitts, O.M., Priest, D.G., Fish, W.W. 1974. Uricase. Subunit composition and resistance to denaturants. Biochem. 13:888–892

    Google Scholar 

  33. Pordy, W.T., Lipkowitz, M.S., Abramson, R.G. 1987. Evidence for the transport fonction of uricase, an oxidative enzyme. Am. J. Physiol. 253:F702-F711

    Google Scholar 

  34. Ran, S., Fuller, C.M., Pia Arrate, M., Latorre, R., Benos, D.J. 1992. Functional reconstitution of a chloride channel protein from bovine trachea. J. Biol. Chem. 267:20630–20637

    Google Scholar 

  35. Sun, X.P., Supplisson, S., Torres, R., Sachs, G., Mayer, E. 1992. Characterization of large-conductance chloride channels in rabbit colonie smooth muscle. J. Physiol. Lond. 448:355–382

    Google Scholar 

  36. Vaca, L., Kunze, D.L. 1992. Anion and cation permeability of a large conductance anion channel in the T84 human colonic cell line. J. Membrane Biol. 130:241–249

    Google Scholar 

  37. Woolley, G.A., Wallace, B.A. 1992. Model ion channels: gramicidin and alamethicin. J. Membrane Biol. 129:109–136

    Google Scholar 

  38. Wu, X., Lee, C.C., Muzny, D.M., Caskey, C.T. 1989. Urate oxidase: primary structure and evolutionary implications. Proc. Natl. Acad. Sci. USA 86:9412–9416

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Renal Division, Department of Medicine, Mount Sinai School of Medicine, 10029, New York, New York

    E. Leal-Pinto, R. D. London, B. A. Knorr & R. G. Abramson

Authors
  1. E. Leal-Pinto
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. R. D. London
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. B. A. Knorr
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. R. G. Abramson
    View author publications

    You can also search for this author in PubMed Google Scholar

Additional information

This work was supported in part by the G. Harold and Leila Y. Mathers Charitable Foundation (E.L.P. and R.D.L.), the Irma T. Hirschl Trust (R.D.L.), National Institutes of Health grant DK08419 (B.A.K.) and a Grant-in-Aid from the American Heart Association, N.Y.C. Affiliate (R.G.A.).

Rights and permissions

Reprints and permissions

About this article

Cite this article

Leal-Pinto, E., London, R.D., Knorr, B.A. et al. Reconstitution of hepatic uricase in planar lipid bilayer reveals a functional organic anion channel. J. Membarin Biol. 146, 123–132 (1995). https://doi.org/10.1007/BF00238003

Download citation

  • Received:

  • Revised:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00238003

Key words

Search

Navigation