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
References
Feb 1, 1975·Proceedings of the National Academy of Sciences of the United States of America·S T ThompsonE Stellwagen
Feb 26, 1990·Biochimica Et Biophysica Acta·Y C YangR H Davis
Feb 10, 1988·Biochimica Et Biophysica Acta·S BeninatiJ E Folk
Jul 1, 1986·Journal of Cellular Physiology·K Y Chen, Z F Chang
Oct 15, 1986·The Biochemical Journal·E W GernerM Siat
Mar 14, 1988·FEBS Letters·K Y Chen, Q P Dou
Aug 19, 1988·Biochimica Et Biophysica Acta·K Y Chen, Q P Dou
Sep 1, 1988·FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology·L M ChenM C Shao
Jul 1, 1974·European Journal of Biochemistry·W M Bonner, R A Laskey
Aug 15, 1970·Nature·U K Laemmli
Jan 1, 1984·Methods in Enzymology·C R Lowe, J C Pearson
Dec 18, 1984·Biochemistry·L CarielloL Lorand
Jan 1, 1984·Annual Review of Biochemistry·C W Tabor, H Tabor
Oct 1, 1984·Experimental Cell Research·B M TorrelioP M Gallop
May 1, 1981·Proceedings of the National Academy of Sciences of the United States of America·M H ParkJ E Folk
Apr 20, 1983·Biochimica Et Biophysica Acta·K Y Chen
Dec 20, 1994·Proceedings of the National Academy of Sciences of the United States of America·J H JacksonC G Cochrane
Nov 2, 1981·FEBS Letters·K Y Chen, A Y Liu
Citations
May 19, 1997·Cancer Letters·Z P Chen, K Y Chen
May 7, 1999·The Biochemical Journal·Y B LeeM H Park
Aug 16, 2000·The EMBO Journal·G LipowskyD Görlich
Oct 15, 2009·Retrovirology·Mainul HoqueMichael B Mathews
Sep 28, 2015·Biochimie·Laura Itzel Quintas-GranadosMaría Elizbeth Álvarez-Sánchez
Mar 1, 1992·European Journal of Biochemistry·D BartigF Klink
Mar 1, 1997·Journal of Cellular Physiology·Z P Chen, K Y Chen
Apr 14, 1995·The Journal of Biological Chemistry·E C WolffM H Park
Jan 6, 1995·The Journal of Biological Chemistry·Y Tao, K Y Chen
Oct 13, 1995·The Journal of Biological Chemistry·Y Tao, K Y Chen