Hypusine-containing proteins and their modifying enzymes

The aim of this study is to purify and characterize this protein and to investigate general properties of its modifying enzymes. I developed a simple four-step procedure for purifying the hyp-18K protein. This procedure resulted in an 800-fold purification of the radio-labeled protein from NB-15 mouse neuroblastoma cells. Two hypusine-containing proteins with an apparent molecular weight of 18 kDa and 20 kDa were also identified by metabolic labeling in chick embryo fibroblasts. The hyp-18K in both NB cells and chick embryo fibroblastes exhibited two isoforms: acidic form (hyp-18Ka) and basic form (hyp-18Kb). I also purified both hyp-20K and hyp-18K isoforms from chick embryos to homogeneity. V8 protease peptide map analysis indicated that hyp-18Kb isolated from chick embryos was identical, while hyp-18Ka and hyp-20K were similar, to elF-4D. The chick embryo hyp-18Kb, but not hyp-18Ka, co-migrated with elF-4D. When NB cell lysates were incubated with [3H]spermidine at pH 9.5, only hyp-18Kb was labeled. Lysates, labeled at pH 9.5 and then adjusted to pH 7.2, gave both hyp-18Ka and hyp-18Kb. These data provided evidence that hyp-18Ka is derived from hyp-18Kb