title: Assay of deoxyhypusine synthase activity.
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-(3)H]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 [(3)H]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 [(3)H]deoxyhypusine-containing eIF5A after separation by SDS-PAGE. The fourth method is a filter-binding assay. It is important to minimize nonspecific binding of [(3)H]spermidine to proteins in the assay mixture, especially for methods 2 and 4, as illustrated in a comparison figure in the chapter.
authors.affiliation: The Oral and Pharyngeal Cancer Branch, NIDCR, National Institutes of Health, Bethesda, MD, USA.
authors.forename: Edith C
authors.initials: EC
authors.lastname: Wolff
authors.forename: Seung Bum
authors.initials: SB
authors.lastname: Lee
authors.forename: Myung Hee
authors.initials: MH
authors.lastname: Park
mesh_terms: D053898:Biosynthetic Pathways; D002457:Cell Extracts; D011232:Chemical Precipitation; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D057075:Enzyme Assays / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D008239:Lysine / Q000031:analogs & derivatives / Q000096:biosynthesis / Q000737:chemistry; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000378:metabolism*; D010448:Peptide Initiation Factors / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism; D011851:Radioactivity; D014238:Trichloroacetic Acid / Q000737:chemistry; D000097574:Eukaryotic Translation Initiation Factor 5A
chemical_list: D002457:Cell Extracts; D008239:Lysine; D000587:Oxidoreductases Acting on CH-NH Group Donors; D010448:Peptide Initiation Factors; D016601:RNA-Binding Proteins; D014238:Trichloroacetic Acid; D000097574:Eukaryotic Translation Initiation Factor 5A; C034924:deoxyhypusine; C063507:deoxyhypusine synthase