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Spermidine mediates degradation of ornithine decarboxylase by a non‐lysosomal, ubiquitin‐independent mechanism
Author(s) -
Glass James R.,
Gerner Eugene W.
Publication year - 1987
Publication title -
journal of cellular physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.529
H-Index - 174
eISSN - 1097-4652
pISSN - 0021-9541
DOI - 10.1002/jcp.1041300119
Subject(s) - spermidine , ornithine decarboxylase , polyamine , putrescine , leupeptin , biochemistry , antipain , ornithine decarboxylase antizyme , chemistry , microbiology and biotechnology , biology , enzyme , protease
Abstract 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 μM exogenous spermidine leads to rapid decay of ODC activity accompanied by a parallel decrease in enzyme protein. Analysis of the decay of [ 35 S]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‐firstorder kinetics over a temperature range of 22–37°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°C for 3 hours with 10 μM 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 (NH 4 Cl, 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°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°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.