Complex formation between deoxyhypusine synthase and its protein substrate, the eukaryotic translation initiation factor 5A (eIF5A) precursor

Deoxyhypusine synthase catalyses the first step in the post-translational synthesis of hypusine [Nepsilon-(4-amino-2-hydroxybutyl) lysine] in a single cellular protein, the precursor of eukaryotic initiation factor 5A (eIF5A). Deoxyhypusine synthase exists as a tetramer with four potential active si...

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Bibliographic Details
Published in:Biochemical journal 1999-05, Vol.340 ( Pt 1) (1), p.273-281
Main Authors: Lee, Y B, Joe, Y A, Wolff, E C, Dimitriadis, E K, Park, M H
Format: Article
Language:English
Subjects:
Cell Extracts
Chromatography, Affinity
deoxyhypusine synthase
Dose-Response Relationship, Drug
Electrophoresis, Polyacrylamide Gel
Escherichia coli - genetics
Eukaryotic Translation Initiation Factor 5A
Guanine - analogs & derivatives
Guanine - pharmacology
Humans
Hydrogen-Ion Concentration
hypusine
initiation factor eIF-5A
Molecular Weight
NAD - pharmacology
Oxidoreductases Acting on CH-NH Group Donors - antagonists & inhibitors
Oxidoreductases Acting on CH-NH Group Donors - metabolism
Peptide Initiation Factors - metabolism
Protein Binding - drug effects
Protein Precursors - metabolism
Protons
Recombinant Fusion Proteins - metabolism
RNA-Binding Proteins
Sequence Analysis
spermidine
Spermidine - pharmacology
Temperature
Thermodynamics
Ultracentrifugation
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Summary:Deoxyhypusine synthase catalyses the first step in the post-translational synthesis of hypusine [Nepsilon-(4-amino-2-hydroxybutyl) lysine] in a single cellular protein, the precursor of eukaryotic initiation factor 5A (eIF5A). Deoxyhypusine synthase exists as a tetramer with four potential active sites. The formation of a stable complex between human deoxyhypusine synthase and its protein substrate, human recombinant eIF5A precursor (ec-eIF5A), was examined by affinity chromatography using polyhistidine-tagged (His.Tag) ec-eIF5A, by a gel mobility-shift method, and by analytical ultracentrifugation. Deoxyhypusine synthase was selectively retained by His.Tag-ec-eIF5A immobilized on a resin. The complex of deoxyhypusine synthase and ec-eIF5A was separated from the free enzyme and protein substrate by electrophoresis under non-denaturing conditions. The stoichiometry of the two components in the complex was estimated to be 1 deoxyhypusine synthase tetramer to 1 ec-eIF5A monomer by N-terminal amino acid sequencing of the complex. Equilibrium ultracentrifugation data further supported this 1:1 ratio and indicated a very strong interaction of the enzyme with ec-eIF5A (Kd
ISSN:0264-6021
1470-8728
DOI:10.1042/0264-6021:3400273