Ornithine decarboxylase (ODC; E.C. is held to be the rate limiting enzyme in the biosynthesis of the polyamines spermidine and spermine, which are essential for cell growth. This enzyme is stringently regulated in all normal cells, and abnormal regulation of ODC has been correlated with tumor formation such that some consider it to be the product of an oncogene. Inhibitors of this biosynthetic pathway are important not only in cancer chemotherapy but also in combating parasitic infections such as the Trypanosomes of African Sleeping Sickness and Pneumocystis carinii associated with AIDS. Although some control of the activity of ODC is exerted at the level of synthesis, this enzyme is most unusual in that it demonstrates an extremely fast protein turnover rate, allowing very rapid modulation of this enzyme activity. This very efficient, specific enzyme deactivation appears to involve an initial enzyme inactivation, which can be stimulated by the products spermidine and spermine, followed quickly by selective degradation of the enzyme protein. We have been investigating post-translational charge modifications associated with phosphate removal from active enzyme states, structural changes in the enzyme, and ODC association with a presumptive regulatory protein, antizyme. Our proposal is to continue these lines of investigation with the intent of assembling this information into a unified model for the controlled deactivation of this enzyme. In particular we will use a rat hepatoma cell line (HTC), kidneys from testosterone- stimulated male mice and adrenal hormone-induced rat tissues to study changes in ODC protein that are produced in vivo. We have developed a non-oxidative, ODC-inactivation system in crude homogenates from HTC cells that will be used to isolate and purify the protease(s) initiating the inactivation/degradation of ODC, and to characterize their substrate specificity and control. Evaluation will also be made of the role of polyamines, antizyme and energy in this rapid, specific protein degradation. The protein phosphatases that convert the more stable ODC isoform to a more labile form will also be isolated, purified and characterized for substrate specificity and control components. It is anticipated that this unified approach to the mechanisms involved in inactivating and degrading this important enzyme will help us understand increases in ODC activity seen in response to various hormones, and to carcinogens and viral transformation, which appear to relate to alterations in this enzyme deactivation process.

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National Institute of General Medical Sciences (NIGMS)
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Biochemistry Study Section (BIO)
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Northern Illinois University
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De Kalb
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Mitchell, J L; Rupert, J; Leyser, A et al. (1998) Mammalian cell polyamine homeostasis is altered by the radioprotector WR1065. Biochem J 335 ( Pt 2):329-34
Mitchell, J L; Judd, G G; Leyser, A et al. (1998) Osmotic stress induces variation in cellular levels of ornithine decarboxylase-antizyme. Biochem J 329 ( Pt 3):453-9
Mitchell, J L; Choe, C Y; Judd, G G (1996) Ornithine decarboxylase stability in HMOA and DH23b cells is not due to post-translational truncation of a C-terminal recognition site. Biochem J 318 ( Pt 3):879-82
Mitchell, J L; Choe, C Y; Judd, G G et al. (1996) Overproduction of stable ornithine decarboxylase and antizyme in the difluoromethylornithine-resistant cell line DH23b. Biochem J 317 ( Pt 3):811-6
Mitchell, J L; Choe, C Y; Judd, G G (1996) Feedback repression of ornithine decarboxylase synthesis mediated by antizyme. Biochem J 320 ( Pt 3):755-60
Mitchell, J L; Judd, G G; Bareyal-Leyser, A et al. (1994) Feedback repression of polyamine transport is mediated by antizyme in mammalian tissue-culture cells. Biochem J 299 ( Pt 1):19-22
Mitchell, J L; Diveley Jr, R R; Bareyal-Leyser, A et al. (1992) Abnormal accumulation and toxicity of polyamines in a difluoromethylornithine-resistant HTC cell variant. Biochim Biophys Acta 1136:136-42
Mitchell, J L; Kurzeja, R J; Marsh, J F et al. (1992) Recovery of ornithine decarboxylase activity after inhibition with alpha-difluoromethylornithine. Biochem Biophys Res Commun 187:443-7
Mitchell, J L; Diveley Jr, R R; Bareyal-Leyser, A (1992) Feedback repression of polyamine uptake into mammalian cells requires active protein synthesis. Biochem Biophys Res Commun 186:81-8
Mitchell, J L; Hoff, J A; Bareyal-Leyser, A (1991) Stable ornithine decarboxylase in a rat hepatoma cell line selected for resistance to alpha-difluoromethylornithine. Arch Biochem Biophys 290:143-52

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