The objective of this research was to identify specific mechanisms involved in the regulation of ornithine decarboxylase, the first enzyme in the polyamine biosynthetic pathway. Immunochemical techniques were used to study post-translational modifications of the ODC protein in relation to activity alterations. Initial experimentation showed that Chinese hamster cells maintained in a defined medium express an ODC protein stable to intracellular degradation. Treatment of these cells with exogenous ornithine or polyamines resulted in a rapid loss of enzyme activity, without detectable changes in the enzyme specific activity. The loss of enzyme activity was a result of accelerated ODC degradation, as determined by immunoprecipitation of pre-labeled protein. In addition, spermidine, but not ornithine, totally inhibited new ODC synthesis. The mechanism of accelerated ODC degradation was investigated and found to occur by an apparent novel mechanism. Degradation of ODC was both ubiquitin-independent and non-lysosomal, and there was also no detectable accumulation of a modified form of ODC protein. In addition, it was found that a component of protein synthesis is required for this process, as inhibitors (cycloheximide, emetine, puromycin) blocked polyamine-accelerated degradation. ODC cDNA was used to synthesize both ODC specific mRNA and protein using in vitro synthesis. These systems may allow the generation of sufficient quantities of material which can be used to recreate in vitro the specific components involved in polyamine inhibition of ODC synthesis and the protease(s) responsible for degradation. The major finding of this work is the direct demonstration that ODC is a stable intracellular protein in the absence of putrescine and spermidine depleted cells (Chapter 2). In addition, that degradation occurs by a novel mechanism, with a requirement for some component of protein synthesis (Chapter 3). Finally, these studies describe the in vitro production of ODC protein and mRNA, which should facilitate further studies of polyamine regulation of ODC degradation and synthesis (Chapter 4).
| Identifer | oai:union.ndltd.org:arizona.edu/oai:arizona.openrepository.com:10150/184002 |
| Date | January 1987 |
| Creators | GLASS, JAMES RUSSELL. |
| Contributors | Gerner, Eugene |
| Publisher | The University of Arizona. |
| Source Sets | University of Arizona |
| Language | English |
| Detected Language | English |
| Type | text, Dissertation-Reproduction (electronic) |
| Rights | Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. |
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