PMID: 2121277Nov 9, 1990Paper

Characterization and reconstitution of a cell free system for NAD(+)-dependent deoxyhypusine formation on the 18 kDa eIF-4D precursor

Biochimica Et Biophysica Acta
Q P Dou, K Y Chen

Abstract

Deoxyhypusine formation on the 18 kDa eIF-4D precursor is due to a covalent linkage between a lysine residue of the protein and the aminobutyl moiety derived from spermidine. The deoxyhypusine is then hydroxylated to form hypusine. This post-translational modification represents one of the most specific spermidine-dependent biochemical events in eukaryotic cells. Deoxyhypusine formation can be performed in vitro at pH 9.5 and is greatly stimulated by NAD+. Using the labeling of the 18 kDa protein by [3H]spermidine as an assay for deoxyhypusine formation, we found that (i) significant deoxyhypusine formation can be demonstrated in vitro at pH 7.2 only if NAD+ is present, (ii) deoxyhypusine formation was sensitive to buffer composition; buffers made of basic amino acids and Tris were inhibitory, (iii) sulfhydryl reagents and metal ions such as Cu2+ and Fe3+ were potent inhibitors of deoxyhypusine formation and (iv) the 18 kDa protein substrate was heat-stable. The in vitro activity of deoxyhypusine formation, which depends on the presence of both enzyme and protein substrate, can be separated from the product, eIF-4D, by a one-step Cibacron blue dye affinity column. Taking advantage of this finding, we have developed a simple pro...Continue Reading

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Citations

Aug 16, 2000·The EMBO Journal·G LipowskyD Görlich
Sep 28, 2015·Biochimie·Laura Itzel Quintas-GranadosMaría Elizbeth Álvarez-Sánchez
Apr 14, 1995·The Journal of Biological Chemistry·E C WolffM H Park

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