Characterization and reconstitution of a cell free system for NAD(+)-dependent deoxyhypusine formation on the 18 kDa eIF-4D precursor.
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 procedure, based on the use of Cibacron blue dye, for partially purifying both the deoxyhypusine-forming enzyme and the 18 kDa protein substrate. When the partially purified enzyme and protein substrate were mixed in the presence of 1 mM NAD+ and [3H]spermidine, the 18 kDa protein was radiolabeled, no labeling could be detected if any one component was absent. Using partially purified enzyme, we have also determined the half-life of the protein substrate in alpha-difluoromethyl ornithine (DFMO)-treated NB-15 cells and found it to be longer than 10 h.
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Department of Chemistry, Rutgers University, New Brunswick, NJ.
Chen
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D000818:Animals; D002460:Cell Line; D002474:Cell-Free System; D008239:Lysine / Q000031:analogs & derivatives* / Q000032:analysis / Q000096:biosynthesis; D051379:Mice; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism*; D009447:Neuroblastoma; D010448:Peptide Initiation Factors / Q000302:isolation & purification / Q000378:metabolism*; D011498:Protein Precursors / Q000302:isolation & purification / Q000378:metabolism*; D016601:RNA-Binding Proteins; D000097574:Eukaryotic Translation Initiation Factor 5A
D008239:Lysine; D009243:NAD; D010448:Peptide Initiation Factors; D011498:Protein Precursors; D016601:RNA-Binding Proteins; D000097574:Eukaryotic Translation Initiation Factor 5A; C100028:hypusine; C034924:deoxyhypusine