Ultrafiltration-based in vitro assay for determining polyamine binding to proteins

doi:10.1038/protex.2012.005

Method Article

Ultrafiltration-based in vitro assay for determining polyamine binding to proteins

https://doi.org/10.1038/protex.2012.005

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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.

Biochemistry

Biological techniques

Computational biology and bioinformatics

ODC

Antizyme

Polyamines

metabolite sensing

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).

Buffer (50 mM Tris-HCl, pH 7.8 at 4°C)
[3H]-spemidine (PerkinElmer, cat No: NET522001MC)
[14C]-spermine (GE Healthcare, cat No: CFA511)
6His-OAZ1 (S. cerevisiae) in IMAC elution buffer (50 mM Tris-HCl, pH 7.8 containing 250 mM imidazole) after expression in E. coli and purification by immobilized metal affinity chromatography (IMAC).
Mock preparation from E. coli cells transformed with an empty vector not expressing 6His-OAZ1.
Scintillation liquid (picard)
Microcentrifuge tubes (Eppendorf)

Refrigerated Microcentrifuge
Centrifugal filter, Modified polyethersulfone (PES) 10K (VWR, Cat No: 82031-350)
Scintillation counter

Keep all reagents on ice. Pre-cool the centrifuge to 4°C.
Label the microcentrifuge tubes and keep them on ice.
Prepare the polyamine binding mix in the pre-cooled microcentrifuge tubes as shown Table A & B shown in figures section.
Mix the polyamine binding mix gently and incubate on ice for 60 min.
Place the filtration device in a 1.5 ml collection tube and pre-cool on ice.
After the 60 min incubation time, transfer 100 μL of the polyamine binding mix to the appropriately labelled filtration devices.
Filter the free polyamines by spinning at 2500xg for 5 min at 4°C
Prepare microcentrifugefuge tubes with 1 mL scintillation liquid (two tubes with scintillation liquid for every filter device used) and keep them at room temperature.
Remove 10 μL of the retentate from inside the cut-off filter device and add it to microcentrifuge tube with scintillation liquid marked as “retentate”.
Remove 10 μL of filtrate from the collection tube of the filtration device and add it to the second microcentrifuge tube with scintillation liquid marked as “filtrate”.
Vortex for 10s and proceed with scintillation counting.
From the resulting CPM (Counts per Minute) values, calculate the percentage of protein-bound polyamines using the following formula.

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.

Binding: CPM in filtrate will be less than retentate
No Binding: CPM in filtrate will be similar to retentate

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.

Wallace, H. M., Fraser, A. V. & Hughes, A. A perspective of polyamine metabolism. Biochem J 376, 1-14 (2003).
Childs, A. C., Mehta, D. J. & Gerner, E. W. Polyamine-dependent gene expression. Cell Mol Life Sci 60, 1394-1406 (2003).
Palanimurugan, R., Scheel, H., Hofmann, K. & Dohmen, R. J. Polyamines regulate their synthesis by inducing expression and blocking degradation of ODC antizyme. Embo J 23, 4857-4867 (2004).
Kurian, L., Palanimurugan, R., Gödderz D., Dohmen R. J. Polyamine sensing by nascent ornithine decarboxylase antizyme stimulates decoding of its mRNA. Nature 477(7365), 490-4 (2011). doi: 10.1038/nature10393.

Authors declare no conflict of interest

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Ultrafiltration-based in vitro assay for determining polyamine binding to proteins

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Ultrafiltration-based in vitro assay for determining polyamine binding to proteins

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Introduction

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Additional Declarations

Associated Publications

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