The processes that control the homeostasis of proteins in cells can discriminate between those proteins within a cell that should be degraded rapidly and those that should not. Highly regulated proteins such as the products of the oncogenes myc and fos, the viral E1a protein, and others whose steady state levels fluctuate in response to various stimuli are invariably short-lived. Understanding, in depth, the processes involved in the degradation of one protein may help elucidate the processes responsible for the homeostasis of other highly regulated and rapidly degraded proteins. ODC is a highly regulated enzyme of the polyamine biosynthetic pathway. It catalyzes the pyridoxal phosphate-dependent decarboxylation of ornithine to putrescine. ODC has been identified as a potential target in the chemotherapy of cancer and parasitic disease because polyamines are essential for growth in all organisms studied. Indeed, a recent paper suggests that ODC is necessary for cell transformation and may fill the requirement of a protooncogene. ODC is known to be very rapidly degraded within cells with a reported half-life ranging from 15 min to 1 hr. The instability of ODC allows enzyme levels to respond promptly to a variety of regulatory mechanisms. Serum, TPA, cAMP and hypotonic shock all increase ODC levels while polyamines at high concentrations cause ODC levels to MI. The induction of ODC is achieved in large part by regulating the rate of degradation. The precise mechanisms by which ODC is constitutively degraded and regulated by polyamine concentrations are still under investigation. Experiments are planned to elucidate the mechanisms responsible for ODC degradation. Specific Aim 1. Specific mutations of ODC will be made and tested for their stability. a. Site-directed mutations converting potential phosphorylation sites to alanines and valines in the C-terminus of ODC will be made and tested for their effects on ODC stability. b. Mutations changing the charge and polarity of the last five amino acids of the carboxyl terminus will be made and tested for their effects on ODC stability. Specific Aim 2. A series of dihydrofolate reductase-ODC chimeras will be made and tested for their ability to be degraded in vivo. Chimeras have the potential of delineating those regions on ODC sufficient for degradation. Biochemical events relevant to degradation will be maintained while others not relevant to degradation will be minimized. Specific Aim 3. How does hypotonic shock inhibit ODC degradation? Is ODC itself modified during hypotonic shock or is the proteolytic machinery affected? Hypotonic shock is known to affect calcium and cAMP levels within the cell. What effects do altering these parameters have on ODC degradation under hypoosmotic and normoosmotic conditions? By analyzing the changes that occur during hypotonic shock, the sequence of events leading to inhibition of ODC degradation may be understood. Specific Aim 4. An in vitro degradation system that reflects the results obtained in vivo will be used to analyze ODC degradation.