Covalent incorporation of polyamines as gamma-glutamyl derivatives into CHO cell protein

Abstract

The possible role of polyamines in the covalent modification of proteins in CHO cells was investigated by metabolic labeling with [3H]putrescine. A single radiolabeled protein band with an apparent relative molecular mass of 18,000 Da was observed by SDS-polyacrylamide gel electrophoresis. Almost all the radioactivity covalently linked to this protein was recovered as hypusine. The labeling of this protein was increased several-fold when cells were cultured with alpha-difluoromethylornithine (DFMO) or with this drug plus methylglyoxal bis(guanylhydrazone) (MGBG), as a result of increase in specific radioactivity of the hypusine immediate precursor, spermidine. Also labeled under the latter condition were other cellular proteins. These were aggregates on the top both of the stacking gel and of the running gel, and protein-like materials with relative molecular masses of 36 and 8 kDa. The radioactivity covalently associated with these proteins was recovered after acid hydrolysis as polyamines. The identification of gamma-glutamylputrescine and gamma-glutamylspermidines in proteolytic digests of the acid-insoluble fraction of treated cells indicates that polyamines are covalently linked to these cellular protein. Several possible cellular functions of gamma-glutamylpolyamine protein components are discussed.