Chen Z P, Chen K Y
Department of Chemistry, Rutgers, The State University of New Jersey, Piscataway 08855-0939, USA.
Cancer Lett. 1997 May 19;115(2):235-41. doi: 10.1016/s0304-3835(97)04741-1.
Hypusine formation on the eukaryotic initiation factor 5A (eIF-5A) precursor is ubiquitously present in eukaryotic cells and archebacteria. In this reaction, deoxyhypusine synthase catalyzes the conversion of one unique lysine residue on eIF-5A to deoxyhypusine using spermidine as the substrate. Hydroxylation of the deoxyhypusine residue completes hypusine formation on eIF-5A. Hypusine formation activity can be measured by an in vitro labeling technique in polyamine-depleted cells. In addition, an in vitro cross-labeling assay can be employed to measure simultaneously the relative deoxyhypusine synthase activity and protein substrate amount. Using these approaches, together with Western blot analysis, we showed that hypusine formation activity is serum-responsive and significantly elevated in Ras oncogene transfected NIH3T3 cells as compared to NIH3T3 cells. The large difference, >30-fold, in hypusine formation activity between these two cells is mainly due to difference in the amount of newly synthesized eIF-5A precursor rather than deoxyhypusine synthase. The deoxyhypusine synthase activity is about three-fold higher in Ras-3T3 cells than in 3T3 cells, and remains constant throughout serum stimulation in both cells. Despite the significant difference in eIF-5A protein amounts, the eIF-5A mRNA levels in 3T3 cells and in Ras-3T3 cells are almost identical. Furthermore, unlike serum-dependent increase in eIF-5A precursor protein, the eIF-5A mRNA in both cells is constitutively expressed after serum stimulation, suggesting that eIF-5A gene is regulated at posttranscriptional/translational level during serum stimulation and cell transformation.
真核起始因子5A（eIF - 5A）前体上的hypusine形成在真核细胞和古细菌中普遍存在。在这个反应中，脱氧hypusine合酶催化eIF - 5A上一个独特的赖氨酸残基以亚精胺为底物转化为脱氧hypusine。脱氧hypusine残基的羟基化完成了eIF - 5A上hypusine的形成。Hypusine形成活性可以通过在多胺缺乏的细胞中进行体外标记技术来测量。此外，可以采用体外交叉标记测定法同时测量相对脱氧hypusine合酶活性和蛋白质底物量。使用这些方法，结合蛋白质印迹分析，我们发现hypusine形成活性对血清有反应，并且与NIH3T3细胞相比，在Ras癌基因转染的NIH3T3细胞中显著升高。这两种细胞之间hypusine形成活性的巨大差异（>30倍）主要是由于新合成的eIF - 5A前体数量的差异，而不是脱氧hypusine合酶的差异。Ras - 3T3细胞中的脱氧hypusine合酶活性比3T3细胞高约三倍，并且在两种细胞的血清刺激过程中保持恒定。尽管eIF - 5A蛋白量存在显著差异，但3T3细胞和Ras - 3T3细胞中的eIF - 5A mRNA水平几乎相同。此外，与血清依赖性增加的eIF - 5A前体蛋白不同，两种细胞中的eIF - 5A mRNA在血清刺激后组成性表达，这表明在血清刺激和细胞转化过程中，eIF - 5A基因在转录后/翻译水平受到调控。
