Interaction of putrescine with nuclear oligopeptides in the enterocyte-like Caco-2 cells

Intestinal cells are able both to synthesize and take up putrescine, the ma in compound of the metabolic poly amine pathway. Polyamine binding to nucle ar macromolecules is thougth to modulate DNA synthesis and transcription. O ur aim was to study the fate of putrescine when taken up from the medium in the enterocyte-like Caco-2 cells and to analyze its binding to nuclear pro teins. After having incubated the cells with C-14-putrescine (0.8 mu M), du ring cell replication and differentiation, the nuclei were separated by seq uential centrifugations in a sucrose gradient. About 20% of the putrescine taken up by Caco-2 cells resulted in the nuclei in both proliferating and d ifferentiated cells. The binding of polyamines to nuclear proteins was stud ied on nuclear extracts, separated by both alkaline polyacrylamide gel elec trophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) and gel pe rmeation chromatography (GPC). No radioactivity was found in nuclear protei n extracts when using the SDS-PAGE method. Conversely, in replicating cells , GPC showed that the greatest amount of radioactivity was present in the n uclear peaks corresponding to oligopeptides with a molecular weight of 4,80 0-8,000 daltons. High-performance liquid chromatography analysis showed the presence of putrescine and spermidine in the 8,000-dalton protein peak, wh ereas in the 4,800-dalton peak spermine was found in addition to putrescine . The radioactive count in the HPLC separated polyamines showed that a smal l percentage of the radioactivity present in the 8,000 and 4,800-dalton GPC peaks was linked to spermine and spermidine, suggesting an interconversion of the supplemented putrescine. Conversely, in differentiated cells, the n uclear oligopeptides did not reveal any radioactivity or any polyamines, su ggesting that the binding of polyamines to nuclear oligopeptides is exclusi vely concerned with replicating cells.