NAD+ stimulated the spermidine-dependent hypusine formation on the 18 kDa protein in cytosolic lysates derived from NB-15 mouse neuroblastoma cells

Abstract

When incubated with cultured mouse neuroblastoma cells under growth stimulatory condition, [3H]putrescine or [3H]spermidine can metabolically label a cellular protein of apparent molecular mass 18 kDa. The labeling, which leads to hypusine formation, is due to a covalent linkage between a lysine residue and the butylamino group derived from spermidine. This reaction can be demonstrated in the cytosolic fractions obtained from cells whose spermidine pool was depleted by prior treatment with alpha-difluoromethylornithine. In an effort to characterize the enzyme system involved in this unique post-translational modification, we found that NAD+ at 0.1 mM stimulated labeling more than 150-fold. Other nucleotides such as NADP+, ATP and GTP were ineffective. The fact that NAD+ dramatically stimulated labeling of the 18 kDa protein indicated that the enzyme involved in hypusine formation may be an NAD+-requiring enzyme.