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

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