, Volume 160, Issue 5, pp 474–479 | Cite as

The effect of solubilisation on the character of an ethylene-binding site from Phaseolus vulgaris L. cotyledons

  • C. J. R. Thomas
  • A. R. Smith
  • M. A. Hall


The ethylene-binding site (EBS) from Phaseolus vulgaris cv. Canadian Wonder cotyledons can be solubilised from 96,000 g pelleted material by Triton X-100 or sodium cholate. Extraction of 96,000 g pellets with acetone, butanol or butanol and ether results in a total loss of ethylene-binding activity. Like the membrane-bound form, the solubilised EBS has an apparent KD(liquid) of 10-10 M at a concentration of 32 pmol EBS per gram tissue fresh weight. Propylene and acetylene act as competitive inhibitors, carbon dioxide appears to promote ethylene binding and ethane has no significant effect. The solubilised EBS is completely denatured affect. The solubilised EBS is completely denatured after 10 min at 70°C, by 1 mM mercaptoethanol and 0.1 mM dithiothreitol, but not by trypsin or chymotrypsin. However, solubilisation decreases the rate constant of association from 103 M-1 s-1 to 101–102 M-1 s-1 and hence does not permit experimental determination of the rate constant of dissociation. The pH optimum for ethylene binding is altered from the range pH 7–10 in the membrane-bound form to the pH range 4–7 in the solubilised form. The EBS appears to be a hydrophobic, intergral membrane protein, which requires a hydrophobic environment to retain its activity. Partitioning of the EBS into polymer phases is determined by the detergent used for solubilisation indicating that when solubilised, the EBS forms a complex with detergent molecules.

Key words

Binding site (ethylene) Ethylene (binding site) Phaseolus (ethylene binding) 



ethylene-binding site


polyethylene glycol


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  1. Albertsson, P.A. (1973) Application of the phase partition method to a hydrophobic membrane protein, phospholipase Al from Escherichia coli. Biochemistry 12, 2525–2530Google Scholar
  2. Bengochea, T., Acaster, M.A., Dodds, J.H., Evans, D.E., Jerie, P.H., Hall, M.A. (1980a) Studies on ethylene binding by cell-free preparations from cotyledons of Phaseolus vulgaris L.: effects of structural analogues of ethylene and inhibitors. Planta 148, 407–411Google Scholar
  3. Bengochea, T., Dodds, J.H., Evans, D.E., Jerie, P.H., Niepel, B., Shaari, A.R., Hall, M.A. (1980b) Studies on ethylene binding by cell-free preparations from cotyledons of Phaseolus vulgaris L: separation and characterisation. Planta 148, 397–406Google Scholar
  4. Burg, S.P., Burg, E.A. (1967) Molecular requirements for the biological activity of ethylene. Plant Physiol. 42, 144–152Google Scholar
  5. Cadman, E., Bostwick, J.R., Eichberg, L. (1979) Determination of protein by a modified Lowry procedure in the presence of some commonly used detergents. Anal. Biochem. 96, 21–23Google Scholar
  6. Cuatrecasas, P. (1972a) Affinity chromatography and purification of the insulin receptor of liver cell membranes. Proc. Natl. Acad. Sci. USA 69, 1277–1281Google Scholar
  7. Cuatrecasas, P. (1972b) Properties of the insulin receptor isolated from liver and fat cell membranes. J. Biol. Chem. 247, 1980–1991Google Scholar
  8. Evans, D.E., Bengochea, T., Cairns, A.J., Dodds, J.H., Hall, M.A. (1982a) Studies on ethylene binding by cell-free preparations of Phaseolus vulgaris L.: subcellular localisation. Plant Cell Environ. 5, 101–107Google Scholar
  9. Evans, D.E., Dodds, J.H., Lloyd, P.C., ap Gwynn, I., Hall, M.A. (1982b) A study of the subcellular localisation of an ethylene binding site in developing cotyledons of Phaseolus vulgaris L. by high resolution autoradiography. Planta 154, 48–52Google Scholar
  10. Ginsberg, B.H., Cohen, R.M., Kahn, C.R., Roth, J. (1978) Properties and partial purification of the detergent solubilised insulin receptor: a demonstration of negative cooperativity in micellar solution. Biochim. Biophys. Acta 542, 88–100Google Scholar
  11. Helenius, A., Simons, K. (1972) The binding of detergents to lipophilic and hydrophilic proteins. J. Biol. Chem. 247, 3656–3661Google Scholar
  12. Helenius, A., Simons, K. (1975) Solubilisation of membranes by detergents. Biochim. Biophys. Acta 415, 29–79Google Scholar
  13. Jerie, P.H., Shaari, A.R., Hall, M.A. (1979) The compartmentation of ethylene in developing cotyledons of Phaseolus vulgaris. Planta 144, 503–507Google Scholar
  14. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275Google Scholar
  15. Maddy, A.H. (1966) The properties of the protein of plasma membrane of ox erythrocytes. Biochim. Biophys. Acta 117, 193–200Google Scholar
  16. Sigrist, H., Sigrist-Nelson, K., Gitler, C. (1977) Single phase butanol extraction: a new tool for proteolipid isolation. Biochem. Biophys. Res. Commun. 74, 178–184Google Scholar
  17. Sisler, E.C. (1980) Partial purification of an ethylene binding component from plant tissue. Plant Physiol. 66, 404–406Google Scholar
  18. Venis, M.A. (1977) Solubilisation and partial purification of auxin-binding sites of corn membranes. Nature (London) 266, 268–269Google Scholar
  19. Wishnia, A. (1962) The solubility of hydrocarbon gases in protein solutions. Proc. Natl. Acad. Sci. USA 48, 2200–2204Google Scholar

Copyright information

© Springer-Verlag 1984

Authors and Affiliations

  • C. J. R. Thomas
    • 1
  • A. R. Smith
    • 2
  • M. A. Hall
    • 2
  1. 1.Biochemistry DepartmentNational Vegetable Research StationWellesbourneUK
  2. 2.Department of Botany and MicrobiologyUniversity College of WalesAberystwythUK

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