Advertisement

The Journal of Membrane Biology

, Volume 133, Issue 1, pp 85–97 | Cite as

Estimate of the number of urea transport sites in erythrocyte ghosts using a hydrophobic mercurial

  • Lidia M. Mannuzzu
  • Mario M. Moronne
  • Robert I. Macey
Articles

Summary

In this paper a variety of mercurials, including a pCMB-nitroxide analogue, were used to study urea transport in human red cell ghosts. It was determined that the rate of inhibition for pCMBS, pCMB, pCMB-nitroxide, and chlormerodrin extended over four orders of magnitude consistent with their measured oil/water partition coefficients. From these results, we concluded that a significant hydrophobic barrier limits access to the urea inhibition site, suggesting that the urea site is buried in the bilayer or in a hydrophobic region of the transporter. In contrast, the rate of water inhibition by the mercurials ranged by only a factor of four and did not correlate with their hydrophobicities. Thus, the water inhibition site may be more directly accessible via the aqueous phase. Under conditions that leave water transport unaffected, we determined that ≤32,000 labeled sites per cell corresponded to complete inhibition of urea transport. This rules out major transmembrane proteins such as band 3, the glucose carrier, and CHIP28 as candidates for the urea transporter. In contrast, this result is consistent with the Kidd (Jk) antigen being the urea transporter with an estimated 14,000 copies per cell. From the experimental number of urea sites, a turnover number between 2–6×106 sec−1 at 22°C is calculated suggesting a channel mechanism.

Key Words

urea transport erythrocytes mercurials spin labels electron spin resonance hydrophobicity 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Belkin, S., Mehlhorn, R.J., Hideg, K., Hankovsky, O., Packer, L., 1987. Reduction and destruction rates of nitroxide spin probes. Arch. Biochem. Biophys. 256(1):232–243Google Scholar
  2. Benga, G., Popescu, O., Pop, V.I. 1986. p-(chloromercuri)benzenesulfonate binding by membrane proteins and the inhibition of water transport in human erythrocytes. Biochemistry 25:1535–1538Google Scholar
  3. Brahm, J. 1977. Temperature-dependent changes of chloride transport kinetics in human red cells. J. Gen. Physiol. 70:283–306Google Scholar
  4. Brahm, J. 1983. Urea permeability of human red cells. J. Gen. Physiol. 82:1–23Google Scholar
  5. Chen, Y.S. 1976. Characterization of rhodopsin sulfhydryl groups in photoreceptor membranes. Ph.D. Thesis, University of California, BerkeleyGoogle Scholar
  6. Colombe, B., Macey, R.I. 1974. Effects of calcium on potassium and water transport in human erythrocyte ghosts. Biochim. Biophys. Acta 363:226–239CrossRefGoogle Scholar
  7. Denker, B.M., Smith, B.L., Kuhajda, F.P., Agre, P. 1988. Identification, purification, and partial characterization of a novel Mr 28,000 integral membrane protein from erythrocytes and renal tubules. J. Biol. Chem. 263(30):15634–15642Google Scholar
  8. Fairbanks, G., Steck, T.L., Wallach, D.F.H. 1971. Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry 10(13):2606–2617Google Scholar
  9. Farmer, E.E.L., Macey, R.I. 1970. Perturbation of red cell volume: Rectification of osmotic flow. Biochim. Biophys. Acta 196:53–65Google Scholar
  10. Finkelstein, A. 1987. Water movement through lipid bilayers, pores and plasma membranes. Theory and reality. Distinguished lecture series of the Society of General Physiologists, Vol. 4. John Wiley & Sons, New YorkGoogle Scholar
  11. Frohlich, O., Macey, R.I., Edwards-Moulds, J., Gargus, J.J., Gunn, R.B. 1991. Urea transport deficiency in Jk(a-b-) erythrocytes. Am. J. Physiol. 260:C778-C783Google Scholar
  12. Frohlich, O., Trammell, D. 1987. Is the red cell urea transporter a carrier or a channel? Biophys. J. 51:514aGoogle Scholar
  13. Funder, J., Wieth, J.O. 1976. Chloride transport in human erythrocytes and ghosts: A quantitative comparison. J. Physiol. 262:679–698Google Scholar
  14. Gargus, J.J., Brunner-Jackson, B., Malone, L. 1988. Cloning the human gene encoding a putative urea transport mechanism. FASEB J. 2:A300Google Scholar
  15. Gargus, J.J., Mitas, M. 1988. Physiological processes revealed through an analysis of inborn errors. Am. J. Physiol. 255:F1047-F1058Google Scholar
  16. Goldstein, D.A., Solomon, A.K. 1960. Determination of equivalent pore radius for human red cells by osmotic pressure measurement. J. Gen. Physiol. 44(1):1–17Google Scholar
  17. Haest, C.W.M., Kamp, D., Deuticke, B. 1981. Topology of membrane sulfhydryl groups in the human erythrocyte. Demonstration of a non-reactive population in intrinsic proteins. Biochim. Biophys. Acta 643:319–326Google Scholar
  18. Heaton, D.C., McLoughlin, K. 1982. Jk(a-b-) red blood cells resist urea lysis. Transfusion 22:70–71Google Scholar
  19. Jay, A.W.L. 1975. Geometry of the human erythrocyte. Biophys. J. 15:205–222Google Scholar
  20. Jones, M.N., Nickson, J.K. 1980. Identifying the monosaccharide transport protein in the human erythrocyte membrane. FEBS Lett. 115(1):1–8Google Scholar
  21. Jones, M.N., Nickson, J.K. 1981. Monosaccharide transport proteins of the human erythrocyte membrane. Biochim. Biophys. Acta 650:1–20Google Scholar
  22. Karan, D.M., Macey, R.I. 1990. Temperatureand concentration-dependence of urea permeability of human erythrocytes determined by NMR. Biochim. Biophys. Acta 1024:271–277Google Scholar
  23. Kedem, O., Katchalsky, A. 1958. Thermodynamic analysis of the permeability of biological membranes to non-electrolytes. Biochim. Biophys. Acta 27:229–246Google Scholar
  24. Knauf, P.A., Rothstein, A. 1971. Chemical modification of membranes. II. Permeation paths for sulfhydryl agents. J. Gen. Physiol. 58:211–223Google Scholar
  25. Levitt, D.G., Mlekoday, H.J. 1983. Reflection coefficient and permeability of urea and ethylene glycol in the human red cell membrane. J. Gen. Physiol. 81:239–253Google Scholar
  26. Lienhard, G.E., Gorga, F.R., Orasky, J.E., Zoccoli, M.A. 1977. Monosaccharide transport system of the human erythrocyte. Identification of the cytochalasin B binding component. Biochemistry 16(22):4921–4926Google Scholar
  27. Lin, S., Spudich, J.A. 1974. Biochemical studies on the mode of action of cytochalasin B. Cytochalasin B binding to red cell membrane in relation to glucose transport. J. Biol. Chem. 249(18):5778–5783Google Scholar
  28. Macey, R.I. 1979. Transport of water and nonelectrolytes across red cell membranes. In: Transport across biological membranes. G. Giebisch, D.C. Tosteson, and H.H. Ussing, editors; pp. 1–57. Springer-Verlag, Berlin, Heidelberg, New YorkGoogle Scholar
  29. Macey, R.I. 1984. Transport of water and urea in red blood cells. Am. J. Physiol. 246:C195-C203.Google Scholar
  30. Macey, R.I. 1986. Mathematical models of membrane transport processes. In: Physiology of Membrane Disorders, T.E. Andreoli, J.F. Hoffman, D.D. Fanestil and S.G. Schultz, editors; pp. 111–131. Plenum, New York, LondonGoogle Scholar
  31. Macey, R.I., Farmer, R.E.L. 1970. Inhibition of water and solute permeability in human red cells. Biochim. Biophys. Acta 211:104–106Google Scholar
  32. Masouredis, S.P., Sudora, E., Mahan, L., Victoria, E.J. 1980. Quantitative immunoferritin microscopy of Fya, Fyb, Jka, U, and Dib antigen site numbers of human red cells. Blood 56(6):969–977Google Scholar
  33. Mayrand, R.R., Levitt, D.G. 1983. Urea and ethylene glycolfacilitated transport systems in the human red cell membrane. Saturation, competition and asymmetry. J. Gen. Physiol. 81:221–237Google Scholar
  34. Mehlhorn, R.J. 1991. Ascorbateand dehydroascorbic acid-mediated reduction of free radicals in the human erythrocyte. J. Biol. Chem. 266(5):2724–2731Google Scholar
  35. Moronne, M.M., Mehlhorn, R.J., Miller, M.P., Ackerson, L.C., Macey, R.I. 1990. ESR measurement of time-dependent and equilibrium volumes of red cells. J. Membrane Biol. 115:31–40Google Scholar
  36. Moura, T.F. 1977. Modifications of red cell permeability by thiol reagents. Ph.D. Thesis, University of California, BerkeleyGoogle Scholar
  37. Moura, T.F., Macey, R.I., Chien, D.Y., Karan, D., Santos, H. 1984. Thermodynamics of all-or-none water channel closure in red cells. J. Membrane Biol. 81:105–111Google Scholar
  38. Mueckler, M., Caruso, C., Baldwin, S.A., Panico, M., Blench, I., Morris, H.R., Allard, W.J., Lienhard, G.E., Lodish, H.F. 1985. Sequence and structure of a human glucose transporter. Science 229:941–945Google Scholar
  39. Naccache, P., Sha'afi, R.I. 1974. Effect of pCMBS on water transfer across biological membranes. J. Cell Physiol. 83(3):449–456Google Scholar
  40. Ojcius, D.M., Solomon, A.K. 1988. Sites of p-chloromercuribenzene sulfonate inhibition of red cell urea and water transport. Biochim. Biophys. Acta 942:73–82Google Scholar
  41. Ojcius, D.M., Toon, M.R., Solomon, A.K. 1988. Is an intact cytoskeleton required for red cell urea and water transport? Biochim. Biophys. Acta 944:19–28Google Scholar
  42. Okubo, Y., Yamaguchi, H., Nagao, N., Tomita, T., Seno, T., Tanaka, M. 1986. Heterogeneity of the phenotype Jk(a-b-) found in Japanese. Transfusion 26:237–239Google Scholar
  43. Preston, G.M., Carroll, T.P., Guggino, W.B., Agre, P. 1992. Appearance of water channels in Xenopus oocytes expressing red cell CHIP28 protein. Science 256:385–387PubMedGoogle Scholar
  44. Sha'afi, R.I., Feinstein, M.B. 1977. Membrane water channels and SH-groups. Adv. Exp. Med. Biol. 84:67–80Google Scholar
  45. Sha'afi, R.I., Rich, T. G., Mikulecky, C. D., Solomon, A.K. 1970. Determination of urea permeability in red cells by minimum method. J. Gen. Physiol. 55:427–450Google Scholar
  46. Shapiro B., Kollmann, G., Martin, D. 1970. The diversity of sulfhydryl groups in the human erythrocyte membrane. J. Cell. Physiol. 75:281–292Google Scholar
  47. Solomon, A.K., Chasan, B., Dix, J.A., Lukacovic, M.F., Toon, M.R., Verkman, A.C. 1983. The aqueous pore in the red cell membrane: Band 3 as a channel for anions, cations, nonelectrolytes and water. Ann. N. Y. Acad. Sci. 414:97–124Google Scholar
  48. Stein, W.D. 1986. Transport and diffusion across cell membranes. Academic, Orlando, FLGoogle Scholar
  49. Toon, M.R., Dorogi, P.L., Lukacovic, M.F., Solomon, A.K. 1985. Binding of DTNB to band 3 in human red cell membrane. Biochim. Biophys. Acta 818:158–170Google Scholar
  50. Toon, M.R., Solomon, A.K. 1986. Control of red cell urea and water permeability by sulfhydryl reagents. Biochim. Biophys. Acta 860:361–375Google Scholar
  51. Toon, M.R., Solomon, A.K. 1987. Interrelation of ethylene glycol, urea and water transport in the red cell. Biochim.Biophys. Acta 898:275–282Google Scholar
  52. VanSteveninck, J., Weed, R.I., Rothstein, A. 1965. Localization of erythrocyte membrane sulfhydryl groups essential for glucose transport. J. Gen. Physiol. 48:617–632Google Scholar
  53. Webb, J.L. 1966. Enzyme and Metabolic Inhibitors. Academic, New YorkGoogle Scholar
  54. Woodfield, D.G., Douglas, R., Smith, J., Simpson, A., Pinder, L., Stavely, J.M. 1982. The Jk(a-b-) phenotype in New Zealand Polynesians. Transfusion 22:276–278Google Scholar
  55. Yousef, L.W., Macey, R.I. 1989. A method to distinguish between pore and carrier kinetics applied to urea transport across the erythrocyte membrane. Biochim. Biophys. Acta 984:281–288Google Scholar

Copyright information

© Springer-Verlag New York Inc. 1993

Authors and Affiliations

  • Lidia M. Mannuzzu
    • 1
  • Mario M. Moronne
    • 2
  • Robert I. Macey
    • 1
  1. 1.Department of Molecular and Cell BiologyUniversity of CaliforniaBerkeley
  2. 2.Life Science DivisionLawrence Berkeley LaboratoryBerkeley

Personalised recommendations