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Polyamines pp 195–205Cite as

Assay of Deoxyhypusine Synthase Activity

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Part of the book series: Methods in Molecular Biology ((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.

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Acknowledgement

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

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Authors and Affiliations

  1. The Oral and Pharyngeal Cancer Branch, NIDCR, National Institutes of Health, Bethesda, MD, USA

    Edith C. Wolff, Seung Bum Lee & Myung Hee Park

Authors
  1. Edith C. Wolff
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  2. Seung Bum Lee
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  3. Myung Hee Park
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Editor information

Editors and Affiliations

  1. College of Medicine, Milton S. Hershey Medical Center, Pennsylvania State University, University Drive 500, Hershey, 17033-0850, Pennsylvania, USA

    Anthony E. Pegg

  2. School of Medicine, Sidney Kimmel Comprehensive, Johns Hopkins University, Orleans St. 1650, Baltimore, 21231, Maryland, USA

    Robert A. Casero, Jr.

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Wolff, E.C., Lee, S.B., Park, M.H. (2011). Assay of Deoxyhypusine Synthase Activity. In: Pegg, A., Casero, Jr., R. (eds) Polyamines. Methods in Molecular Biology, vol 720. Humana Press. https://doi.org/10.1007/978-1-61779-034-8_12

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  • DOI: https://doi.org/10.1007/978-1-61779-034-8_12

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  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-033-1

  • Online ISBN: 978-1-61779-034-8

  • eBook Packages: Springer Protocols

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