Abstract
Binding proteins, thought to be auxin receptors, can be solubilised from maize (Zea mays L.) membranes after acetone treatment. From these crude extracts, receptor preparations of over 50% purity can be obtained by a reliable, straight-forward procedure involving three chromatographic steps — anion exchange, gel filtration and high-resolution anion exchange. Such preparations have been used to immunise rats for subsequent production of monoclonal antibodies. By the further step of native polyacrylamide gel electrophoresis the semi-purified preparations yield homogeneous, dimeric (22-kilodalton, kDa) auxin-binding protein, which has been used to produce a polyclonal rabbit antiserum. The preliminary characterisation of this antiserum and of the five monoclonal antibodies is presented. Two of the monoclonal antibodies specifically recognise the major 22-kDa-binding protein polypeptide whilst the other three recognise, in addition, a minor 21-kDa species. All the monoclonal antibodies recognise the polypeptide rather than the glycan side chain and the polyclonal antiserum also recognises deglycosylated binding protein. The antibodies have been used to quantify the abundance of auxinbinding protein in a number of tissues of etiolated maize seedlings. Root membranes contain 20-fold less binding protein than coleoptile membranes.
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Abbreviations
- ABP:
-
auxin-binding protein
- DEAE:
-
diethylaminoethyl
- Ig:
-
immunoglobulin
- kDa:
-
kilodalton
- NAA:
-
naphthalene-1-acetic acid
- Mr :
-
relative molecular mass
- PAGE:
-
polyacrylamide gel electrophoresis
- SDS:
-
sodium dodecyl sulfate
References
Batt, S., Wilkins, M.B., Venis, M.A. (1976) Auxin binding to corn coleoptile membranes: kinetics and specificity. Planta 130, 7–13
Bazin, H. (1982) Production of rat monoclonal antibodies with the LOU rat non-secreting IR983F myeloma cell line. In: Protides of the biological fluids, 29th Colloquium 1981, pp. 615–618, Peeters, H., ed., Pergamon Press, Oxford New York
Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein-dye binding. Anal. Biochem. 72, 248–254
Dunn, S.D. (1986) Effects of the modification of transfer buffer composition and the rematuration of proteins in gells on the recognition of proteins on Western blots by monoclonal antibodies. Anal. Biochem. 157, 144–153
Faye, L., Chispeels, M.J. (1985) Characterisation of N-linked oligosaccharides by affinoblotting with concanavalin A-peroxidase and treatment of the blots with glycosidases. Anal. Biochem. 149, 218–224
Foung, S.K.H., Sasaki, D.T., Grumet, F.C., Engleman, E.G. (1982) Production of functional human T-T hybridomas in selection medium lacking aminopterin and thymidine. Proc. Natl Acad. Sci. USA 79, 7474–7488
Galfrè, G., Milstein, C. (1981) Preparation of monoclonal antibodies. Strategies and procedures. Methods Enzymol. 73, 3–46
Hames, B.D. (1981) An introduction to polyacrylamide gel electrophoresis. In: Gel electrophoresis of proteins: a practical approach, pp. 1–91, Hames, B.D., Rickwood, D., eds. IRL Press, London Washington
Hertel, R., Thomson, K-St., Russo, V.E.A. (1972) In vitro auxin binding to particulate cell fractions from corn coleoptiles. Planta 107, 325–340
Kumar, B.V., Lakshmi, M.V., Atkinson, J.P. (1985) Fast and efficient method for detection and estimation of proteins. Biochem. Biophys. Res. Commun 131, 883–891
Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680–685
Löbler, M., Klämbt, D. (1985) Auxin-binding protein from coleoptile membranes of corn. I. Purification by immunological methods. J. Biol. Chem. 260, 9848–9853
Löbler, M., Simon, K., Hesse, T., Klämbt, D. (1987) Auxin receptors in target tissue. In: Molecular biology of plant growth control, pp. 279–288, Fox, J.E., Jacobs, M., eds, Alan R. Liss, New York
Murphy, G.J.P. (1980) Naphthaleneacetic acid and binding by membrane-free preparations of cytosol from the maize coleoptile. Plant Sci. Lett. 19, 157–168
Notton, B.A., Fido, R.J., Galfrè, G. (1985) Monoclonal antibodies to a higher plant nitrate reductase: differential inhibition of enzyme activities. Planta 165, 114–119
Ray, P.M., Dohrmann, U., Hertel, R. (1977) Specificity of auxin-binding sites on maize coleoptile membranes as possible receptor sites for auxin action. Plant Physiol. 60, 585–591
Shimomura, S., Sotobayashi, T., Futai, M., Fukui, T. (1986) Purification and properties of an auxin-binding protein from maize shoot membranes. J. Biochem. 99, 1513–1524
Tappeser, B., Wellnitz, D., Klämbt, D. (1981) Auxin affinity proteins prepared by affinity chromatography. Z. Pflanzenphysiol. 101, 295–302
Tarentino, A.L., Maley, F. (1974) Purification and properties of an endo-β-N-acetylglucosaminidase from Streptomyces griseus. J. Biol. Chem. 249, 811–817
Venis, M.A. (1977) Solubilisation and partial purification of auxin-binding sites of corn membranes. Nature (London) 66, 268–269
Venis, M.A. (1980) Purification and properties of membranebound auxin receptors in corn. In: Plant growth substances 1979, pp. 61–70, Skoog, F. ed. Springer, Berlin Heidelberg New York
Venis, M.A. (1984) Hormone-binding studies and the misuse of precipitation assays. Planta 162, 502–505
Venis, M.A. (1985) Hormone binding sites in plants. Longman, New York London
Venis, M.A. (1987) Can auxin receptors be purified by affinity chromatography? In: Plant hormone receptors (Proc. NATO Advanced Workshop, Bonn, FRG, 1986), pp. 27–39, Klämbt, D. ed. Springer, Berlin Heidelberg New York
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Napier, R.M., Venis, M.A., Bolton, M.A. et al. Preparation and characterisation of monoclonal and polyclonal antibodies to maize membrane auxin-binding protein. Planta 176, 519–526 (1988). https://doi.org/10.1007/BF00397659
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DOI: https://doi.org/10.1007/BF00397659