Mitchell, John L. A.
M.S. (Master of Science)
Department of Biological Sciences
The rate-limiting enzyme of the polyamine biosynthetic pathway in mammalian cells is ornithine decarboxylase (ODC; E.C. 220.127.116.11). In rat hepatoma (HTC) cells, ODC exists in two distinct charged forms that are separable on an anion exchange column. Perhaps the most distinctive characteristic of ODC is that it has possibly the shortest half-life of any mammalian enzyme. One of the most fundamental but poorly understood aspects of ODC is which product of the polyamine biosynthetic pathway is responsible for feedback control. In this study I have attempted to answer this question primarily by limiting the production of the polyamines through the inhibition of the enzymes in the pathway. Methylglyoxyl bis(guanlyhydrazone) (MGBG) which inhibits S-adenosylmethionine decarboxylase and dicyclohexylamine (DCHA) which inhibits spermidine synthase were used to selectively deplete the polyamines. By examining the levels in cells treated with these inhibitors and determining the activity of ODC, spermidine appeared responsible for ODC inactivation. Exogenous addition of spermidine to cell cultures, depleted of the polyamines, resulted in a rapid turnover of the enzyme. Using cycloheximide spermidine was discovered to induce the synthesis of a degradative protein which caused the short half-life of ODC. In addition to turnover of total ODC activity, the half-lives of the two forms of ODC were examined. When ODC activity is high due to spermidine depletion, and the enzyme half-life is very long, ODC-II is 3.5-fold more stable than ODC-I. With the addition of spermidine, the synthesis of the degradative protein is induced and ODC-I and II prove equally susceptible to the rapid degradation. This suggests that two mechanisms of ODC inactivation exist in mammalian cells.
Mahan, Dennis W., "The effects of polyamines on the control of ODC in mammalian cells" (1985). Graduate Research Theses & Dissertations. 5683.
vi, 71 pages
Northern Illinois University
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