Abstract
Studies on hormone-receptor interaction include, as a rule, isolation and extensive purification of the receptor protein or a particular receptor-containing fraction. To bypass these time- and resource-consuming procedures, we proposed a live cell-based assay using transgenic bacteria expressing single eukaryotic receptors. We describe here 3H-cytokinin binding to corresponding plant receptors as an example. The method includes procedures of bacteria growing, incubation with labeled hormone, separation of bound from unbound ligand, determination of radioactivity in bacterial precipitates, and mathematical analysis of primary data. The established simple protocol for specific labeling hormone-binding sites in intact bacteria allows determination of the main parameters of the ligand–receptor interaction.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Gomperts, B.D., Kramer, I.M., and Tatham, P.E.R. (2003) Signal Transduction. Academic Press, San Diego et al.
Cuatrecasas, P., and Jacobs, S. (eds.) (1981) Membrane Receptors, Methods for Purification and Characterization. Chapman and Hall, London, N.Y.
Muramatsu, I., Tanaka, T., Suzuki, F., Li, Z., Hiraizumi-Hiraoka, Y., Anisuzzaman, A.S.M., Yamamoto, H., Horinouchi, T., and Morishima, S. (2005) Quantifying receptor properties: the tissue segment binding method – a powerful tool for the pharmacome analysis of native receptors. J. Pharmacol. 98, 331–339.
Suzuki, T., Miwa, K., Ishikawa, K., Yamada, H., Aiba, H., and Mizuno, T. (2001) The Arabidopsis sensor His-kinase, AHK4, can respond to cytokinins. Plant Cell Physiol. 42, 107–113.
Yamada, H., Suzuki, T., Terada, K., Takei, K., Ishikawa, K., Miwa, K., Yamashino, T., and Mizuno, T. (2001) The Arabidopsis AHK4 histidine kinase is a cytokinin-binding receptor that transduces cytokinin signals across the membrane. Plant Cell Physiol. 42, 1017–1023.
SpĂchal, L., Rakova, N.Y., Riefler, M., Mizuno, T., Romanov, G.A., Strnad, M., and SchmĂĽlling, T. (2004) Two cytokinin receptors of Arabidopsis thaliana, CRE1/AHK4 and AHK3, differ in their ligand specificity in a bacterial assay. Plant Cell Physiol. 45, 1299–1305.
Romanov, G.A., SpĂchal, L., Lomin, S.N., Strnad, M., and SchmĂĽlling, T. (2005) A live cell hormone-binding assay on transgenic bacteria expressing a eukaryotic receptor protein. Analyt. Biochem. 347, 129–134.
Romanov, G.A., Lomin, S.N., and Schmülling, T. (2006) Biochemical characteristics and ligand-binding properties of Arabidopsis cytokinin receptor AHK3 compared to CRE1/AHK4 as revealed by a direct binding assay. J. Exp. Botany, 57, 4051–4058.
Heyl, A., Wulfetange, K., Pils, B., Nielsen, N., Romanov, G.A., and SchmĂĽlling, T. (2007) Evolutionary proteomics identifies amino acids essential for ligand-binding of the cytokinin receptor CHASE domain. BMC Evolutionary Biol. 7, 62
Sambrook, J., and Russell, D.W. (2001) Molecular Cloning: a Laboratory Manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
Lomin S.N., and Romanov, G.A. (2008) The analysis of hormone-receptor interaction. Theoretical and practical aspects. Russian J. Plant Physiol. 55, 259–273
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Romanov, G.A., Lomin, S.N. (2009). Hormone-Binding Assay Using Living Bacteria Expressing Eukaryotic Receptors. In: Cutler, S., Bonetta, D. (eds) Plant Hormones. Methods in Molecular Biology, vol 495. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-59745-477-3_10
Download citation
DOI: https://doi.org/10.1007/978-1-59745-477-3_10
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-934115-32-9
Online ISBN: 978-1-59745-477-3
eBook Packages: Springer Protocols