DEOXYHYPUSINE SYNTHASE FROM RAT TESTIS - PURIFICATION AND CHARACTERIZATION

Deoxyhypusine synthase is the first enzyme involved in the post transl ational formation of hypusine, a unique amino acid that occurs at one position in a single cellular protein, eukaryotic translation initiati on factor 5A (eIF-5A). This NAD-dependent enzyme catalyzes the formati on of deoxyhypusine by transfer of the butylamine portion of spermidin e to the epsilon-amino group of a specific lysine residue in the eIF-5 A precursor. Its purification from rat testis was accomplished by ammo nium 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 sequenc es of five tryptic peptides of the pure enzyme did not correspond to a ny sequences in the protein data banks. The enzyme migrates as a singl e band ton SDS-polyacrylamide gel electrophoresis with an apparent mon omer molecular mass of similar to 42,000 Da. Matrix-assisted laser des orption mass spectrometry gave a monomer mass of 40,800 Da. There is e vidence, however, that the active enzyme exists as a tetramer of this subunit. Rabbit polyclonal antibodies to the 42-kDa protein precipitat ed deoxyhypusine synthase activity. The enzyme shows a strict specific ity for NAD. Purified deoxyhypusine synthase catalyzes the overall syn thesis of deoxyhypusine and, in the absence of the eIF-5A precursor, c atalyzes the cleavage of spermidine.