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Polyamines pp 195-205 | Cite as

Assay of Deoxyhypusine Synthase Activity

  • Edith C. Wolff
  • Seung Bum Lee
  • Myung Hee Park
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 720)

Abstract

Deoxyhypusine synthase catalyzes an unusual protein modification reaction. A portion of spermidine is covalently added to one specific lysine residue of one eukaryotic protein, eIF5A (eukaryotic initiation factor 5A) to form a deoxyhypusine residue. The assay measures the incorporation of radioactivity from [1,8-3H]spermidine into the eIF5A protein. The enzyme is specific for the eIF5A precursor protein and does not work on short peptides (<50 amino acids). Optimum conditions for the reaction and four detection methods for the product, deoxyhypusine-containing eIF5A, are described in this chapter. The first, and most specific, method is the measurement of the amount of [3H]deoxyhypusine in the protein hydrolysate after its separation by ion exchange chromatography. However, this method requires some specialized equipment. The second method is counting the radioactivity in TCA-precipitated protein after thorough washing. The third method involves determining the radioactivity in the band of [3H]deoxyhypusine-containing eIF5A after separation by SDS-PAGE. The fourth method is a filter-binding assay. It is important to minimize nonspecific binding of [3H]spermidine to proteins in the assay mixture, especially for methods 2 and 4, as illustrated in a comparison figure in the chapter.

Key words

Polyamine Spermidine Deoxyhypusine synthase Deoxyhypusine Hypusine eIF5A Posttranslational modification 

Notes

Acknowledgement

This research was supported by the Intramural Research Program of the NIDCR, National Institutes of Health.

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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Edith C. Wolff
    • 1
  • Seung Bum Lee
    • 1
  • Myung Hee Park
    • 1
  1. 1.The Oral and Pharyngeal Cancer BranchNIDCR, National Institutes of HealthBethesdaUSA

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