Polyamines (spermidine and spermine) and putrescine are essential for cell growth and function. To understand the molecular basis of the action of polyamines in the regulation of growth and differentiation, it is important to focus on specific polyamine-dependent biochemical events. Hypusine formation in an 18kDa cellular protein is by far the most specific polyamine-dependent biological process discovered. This reaction represents a unique post-translational modification in which the aminobutyl moiety of spermidine is transferred to the epsilon-amino group of a lysine residue of an 18kDa protein (catalyzed by E1). This modification appears to be highly conserved, present in fungus as well as all mammalian cells. Our previous studies indicate that hypusine formation can be stimulated by serum and is diminished during mouse neuroblastoma differentiation. The specificity, ubiquity, and its association with cell proliferation suggest that hypusine formation may be of fundamental importance in cell growth regulation. It is possible that some of the important physiological functions of polyamines are mediated via hypusine formation in the 18kDa protein. We have recently found that NAD+ at 0.1 to 1 mM dramatically stimulates hypusine formation in cytosolic lysates isolated from mouse neuroblastoma cells. We also found that the substrate protein in Neurospora crassa has an apparent molecular weight of 21kDa and that neuroblastoma E1 can use the 21kDa as a substrate. These findings allow us to develop both purification and assay procedures for E1 and 18kDa protein. In this proposal, we will focus on the purification and characterization of E1 and 18kDa. Purified proteins will be used to develop enzymatic assay procedure, to produce antibodies, and to isolate cDNA clones for E1 and 18kDa. The enzymatic assay and various probes generated will be used to study the regulation of these proteins in mouse neuroblastoma cells during growth and differentiation. We have previously shown that the differentiation of mouse neuroblastoma cells is accompanied by a significant decrease of spermidine content. Since hypusine formation is solely spermidine dependent, it is possible that regulation of hypusine formation may have a role in mediating the differentiation of mouse neuroblastoma cells.
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