Deoxyhypusine synthase from rat testis: purification and characterization.
Deoxyhypusine synthase is the first enzyme involved in the post-translational formation of hypusine, a unique amino acid that occurs at one position in a single cellular protein, eukaryotic translation initiation factor 5A (eIF-5A). This NAD-dependent enzyme catalyzes the formation of deoxyhypusine by transfer of the butylamine portion of spermidine to the epsilon-amino group of a specific lysine residue in the eIF-5A precursor. Its purification from rat testis was accomplished by ammonium sulfate fractionation and successive ion-exchange chromatographic steps, followed by chromatofocusing on a hydrophilic resin (Mono P). A pI of 4.7 was determined by isoelectric focusing. Amino acid sequences of five tryptic peptides of the pure enzyme did not correspond to any sequences in the protein data banks. The enzyme migrates as a single band on SDS-polyacrylamide gel electrophoresis with an apparent monomer molecular mass of approximately 42,000 Da. Matrix-assisted laser desorption mass spectrometry gave a monomer mass of 40,800 Da. There is evidence, however, that the active enzyme exists as a tetramer of this subunit. Rabbit polyclonal antibodies to the 42-kDa protein precipitated deoxyhypusine synthase activity. The enzyme shows a strict specificity for NAD. Purified deoxyhypusine synthase catalyzes the overall synthesis of deoxyhypusine and, in the absence of the eIF-5A precursor, catalyzes the cleavage of spermidine.
Wolff
E C
EC
Enzyme Chemistry Section, National Institute of Dental Research, NIH, Bethesda, Maryland 20892-4330, USA.
Lee
Y B
YB
Chung
S I
SI
Folk
J E
JE
Park
M H
MH
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000906:Antibodies / Q000276:immunology; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D008297:Male; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009711:Nucleotides / Q000378:metabolism; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D013379:Substrate Specificity; D013737:Testis / Q000201:enzymology*
D000596:Amino Acids; D000906:Antibodies; D009711:Nucleotides; D000587:Oxidoreductases Acting on CH-NH Group Donors; C063507:deoxyhypusine synthase