Deoxyhypusine synthase assay based on the use of polyhistidine-tagged substrate and metal chelate-affinity chromatography.
Hypusine formation on the eukaryotic initiation factor 5A (eIF-5A) precursor is a unique spermidine-dependent post-translational modification that appears to be ubiquitous in eukaryotic cells. In this modification, a specific lysine residue of eIF-5A precursor protein is first converted to deoxyhypusine by an addition of a butylamino group derived from spermidine; the deoxyhypusine residue is then hydroxylated to form hypusine. Deoxyhypusine synthase, an NAD(+)-dependent enzyme, catalyzes the first step of hypusine formation on the eIF-5A precursor. Since deoxyhypusine formation represents one of the most specific polyamine-dependent reactions in eukaryotic cells, the reaction may play an important role in cellular growth regulation. To facilitate the study of the function and significance of deoxyhypusine formation, we have developed a rapid and sensitive assay for deoxyhypusine synthase. The assay relied on the use of hexahistidine-tagged recombinant Neurospora 21-kDa eIF-5A precursor protein as the substrate protein. The radiolabeled polyhistidine-tagged protein, once modified by [3H]spermidine, was separated from free [3H]spermidine by a microscale metal-affinity chromatography in a dot blot filtration apparatus and quantified by liquid scintillation counting. The assay procedure is quick and simple compared to other methods reported in the literature. The sensitivity is limited by the specific activity of [3H]-spermidine in the reaction mixture. The metal-affinity chromatographic assay for deoxyhypusine synthase should facilitate the purification, characterization, and functional studies of this enzyme.
Tao
Y
Y
Department of Chemistry, Rutgers, State University of New Jersey, Piscataway 08855-0939.
Skrenta
H M
HM
Chen
K Y
KY
D002846:Chromatography, Affinity; D006639:Histidine; D007700:Kinetics; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000032:analysis*; D010455:Peptides / Q000378:metabolism; D013095:Spermidine / Q000378:metabolism*
D010455:Peptides; C033223:polyhistidine; D006639:Histidine; D000587:Oxidoreductases Acting on CH-NH Group Donors; C063507:deoxyhypusine synthase; D013095:Spermidine