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 α-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.