Polyamine conjugation during the cell cycle of Helianthus tuberosus: non enzymatic and transglutaminase-like binding activity

Parenchyma cells of Helianthus tuberosus tuber slices were activated with 2,4-dichlorophenoxyacetic acid in order to induce a synchronous cell cycle. In vivo polyamine conjugation during the cell cycle was studied by activating the slices in a medium containing [14C]putrescine. In vitro polyamine conjugates were obtained by incubating activated slice extracts with both [3H]- and [14C]putrescine as tracers. Different methods of isolation of the conjugates were assayed. Two different putrescine binding activities have been found: a temperature-sensitive one (possibly a transglutaminase-like activity) which, after an early decrease, rose in late G1-early S phase and then remained constant, and a temperature-insensitive activity which slowly increased during the cycle. A thin layer chromatography analysis, after acid hydrolysis, of the in vitro conjugates showed that the main bound polyamine was putrescine; other derivatives, in different percentages according to the type of labelled precursor used, were also found. Sodium dodecylsulphate polyacrylamide gel electrophoresis fluorograms showed that with both labels, heavy molecular weight conjugates were obtained both in vivo and in vitro that cannot enter the running gel; they were more stable when produced by the temperature-sensitive activity. Many [14C]putrescine conjugates of lower molecular weight were also found by the in vitro assay.