Spermidine mediates degradation of ornithine decarboxylase by a non-lysosomal, ubiquitin-independent mechanism.

The mechanism of spermidine-induced ornithine decarboxylase (ODC, E.C. 4.1.1.17) inactivation was investigated using Chinese hamster ovary (CHO) cells, maintained in serum-free medium, which display a stabilization of ODC owing to the lack of accumulation of putrescine and spermidine (Glass and Gerner: Biochem. J., 236:351-357, 1986; Sertich et al.: J. Cell Physiol., 127:114-120, 1986). Treatment of cells with 10 microM exogenous spermidine leads to rapid decay of ODC activity accompanied by a parallel decrease in enzyme protein. Analysis of the decay of [35S]methionine-labeled ODC and separation by two-dimensional electrophoresis revealed no detectable modification in ODC structure during enhanced degradation. Spermidine-mediated inactivation of ODC occurred in a temperature-dependent manner exhibiting pseudo-first-order kinetics over a temperature range of 22-37 degrees C. In cultures treated continuously, an initial lag was observed after treatment with spermidine, followed by a rapid decline in activity as an apparent critical concentration of intracellular spermidine was achieved. Treating cells at 22 degrees C for 3 hours with 10 microM spermidine, followed by removal of exogenous polyamine, and then shifting to varying temperatures, resulted in rates of ODC inactivation identical with that determined with a continuous treatment. Arrhenius analysis showed that polyamine mediated inactivation of ODC occurred with an activation energy of approximately 16 kcal/mol. Treatment of cells with lysosomotrophic agents (NH4Cl, chloroquine, antipain, leupeptin, chymostatin) had no effect on ODC degradation. ODC turnover was not dependent on ubiquitin-dependent proteolysis. Shift of ts85 cells, a temperature-sensitive mutant for ubiquitin conjugation, to 39 degrees C (nonpermissive for ubiquitin-dependent proteolysis) followed by addition of spermidine led to a rapid decline in ODC activity, with a rate similar to that seen at 32 degrees C (the permissive temperature). In contrast, spermidine-mediated ODC degradation was substantially decreased by inhibitors of protein synthesis (cycloheximide, emetine, and puromycin). These data support the hypothesis that spermidine regulates ODC degradation via a mechanism requiring new protein synthesis, and that this occurs via a non-lysosomal, ubiquitin-independent pathway.