scientificprotocols authored about 6 years ago
Authors: Palanimurugan R & R Jürgen Dohmen
We have established an ultrafiltration-based in vitro assay for determining polyamine binding to proteins. In the protocol described here we have used Saccharomyces cerevisiae ODC antizyme protein produced in E. coli and radioactive polyamines to measure the direct binding of polyamines to ODC antizyme protein.
Polyamines are small aliphatic polycations present in all cells. Polyamines are known to have several important functions, which are essential for life (Ref. 1). The cellular polyamine pool contains several modified forms of polyamines, however putrescine, spemidine and spermine are the most abundant among others. Normally polyamines exert their molecular functions by direct binding to cellular macromolecules such as DNA, RNA and proteins (Refs. 1 and 2). We have established an in vitro polyamine binding assay that enables identification and characterization of novel polyamine targets. Using this ultrafiltration-based protocol, we have assayed polyamine (spermidine or spermine) binding to yeast or human ornithine decarboxylase (ODC) antizyme (AZ) (Refs. 3 and 4) protein produced in Escherichia coli. Apart from being easy and fast, this protocol requires only small quantities of purified proteins for determination of polyamine binding. Combined with protein fractionation methods, this protocol can be applied for the identification of novel intra or extra cellular polyamine binding proteins. The protocol given below describes determination of polyamine binding for 6His-tagged S. cerevisiae ODC antizyme (OAZ1). An adopted version of this protocol was used to determine polyamine binding to human antizyme fused to maltose binding protein (Ref. 4).
Percentage polyamine Binding = {(CPMretentate- CPMfiltrate)/ CPMretentate} x 100
About 2 hours
No Binding: Verify that the protein did not precipitate during the assay. Perform the polyamine binding assay immediately after purification of protein. Strictly avoid freezing and thawing of purified OAZ1.
Representative results for spermine and spermidine binding to S. cerevisiae OAZ1 are shown in Fig. 4 and Supplementary Fig. 9 of Kurian et al. Nature, 2011, Doi: 10.1038/nature10393.
Table A & B: Radio-labeled polyamine binding mix
Polyamine sensing by nascent ornithine decarboxylase antizyme stimulates decoding of its mRNA. Leo Kurian, R. Palanimurugan, Daniela Gödderz, and R. Jürgen Dohmen. Nature 477 (7365) 490 - 494 doi:10.1038/nature10393
Palanimurugan R & R Jürgen Dohmen, Biocenter, Institute for Genetics, University of Cologne, Zülpicher Str. 47a, D-50674, Cologne, Germany
Correspondence to: Palanimurugan R ([email protected])
Source: Protocol Exchange (2012) doi:10.1038/protex.2012.005. Originally published online 29 February 2012.