Abstract Ornithine decarboxylase (EC 4.1.1. 17) is the first and the rate-controlling enzyme in polyamine biosynthesis. It decarboxylates L-ornithine to form diamine putrescine. ODC activity in cells is strictly regulated and one of the central elements of ODC regulation is an inhibitory protein called antizyme. Antizyme binds to ODC, inhibits its activity and targets ODC for the proteasomal degradation. Essentiality of polyamines for the normal cell growth and proliferation is well known. Recently their roles in the regulation of several classes of cation channels have been discovered. Some of these channels are expressed abundantly in the brain, which has increased interest in the polyamine metabolism in the central nervous system. In this study guanosine 5'-triphosphate activatable ODC was detected in the rat brain lysates. This activation was more significant after antizyme was separated from ODC. GTP-activatable ODC was more resistant to heat and displayed higher Vmax than kidney ODC. Previously GTP-activatable ODC had been found in mammalian tissues only in some tumors. ODC and antizyme expression in brain was localized by in situ hybridization and immunocytochemistry. Both proteins displayed wide and largely overlapping expression patterns restricted to neurons. The proteins were localized predominantly to cytoplasm at the most brain regions, but antizyme had a main localization in nuclei in some regions of the brain. In addition, the role of one of the most highly conserved regions in eukaryotic ODCs was studied using site-directed mutagenesis. The aspartate-233 to valine mutation was made and detected to increase Km values for the cofactor PLP and the substrate L-ornithine as well as Ki value for the inhibitor DFMO. In another part of this study a transgenic mouse line expressing ODC under the control of viral promotor was generated. The most significant changes in ODC activity were detected in reproductive organs of male mice. The high number of infertile transgenic males supported earlier reports about the importance of balanced polyamine metabolism for spermatogenesis. Infertility of female mice was increased as well, but the involvement of polyamines remained unproven. Transgenic mice were prone to various pathological conditions such as inflammations and tumour formation, which may be due to deregulated polyamine metabolism.

Las poliaminas regulan procesos de crecimiento y diferenciación celular, y su desregulación está relacionada con diferentes patologías incluyendo el cáncer. Las antizimas (AZs) de ornitina descarboxilasa (ODC) inhiben tanto su biosíntesis, como su captación, regulando los niveles intracelulares de poliaminas. En esta tesis se ha caracterizado una nueva proteína inhibidora de antizimas (AZIN2) que posee alta homología con ODC y el inhibidor de antizimas previamente conocido (AZIN1). Esta nueva proteína está desprovista de actividad enzimática, pero es capaz de revertir la acción que las tres antizimas conocidas ejercen sobre la actividad ODC y la captación de poliaminas. A diferencia de sus proteínas homólogas, AZIN2 se localiza subcelularmente en el ERGIC, y se expresa específicamente en cerebro y testículo, pero de forma muy abundante en espermátidas y espermatozoides, al igual que AZ3, indicando que estas dos proteínas juegan un importante papel regulando los niveles de poliaminas durante la espermiogénesis. / Polyamines regulate cell growth and differentiation, and the alteration of their homeostasis is related to different diseases, including cancer. Ornithine decarboxylase (ODC) antizymes (AZs) regulate polyamine levels by inhibiting both their biosynthesis and the cellular uptake. In this work, a new ODC paralogue has been characterized as a novel antizyme inhibitor protein that has been named AZIN2. This protein lacks decarboxylating activity, but it is able to reverse the action of any of the three antizymes on ODC activity and polyamine uptake. Unlike its homologue proteins ODC and AZIN1, AZIN2 is located in the ERGIC, and it is specifically expressed in brain and testes. The abundant expression in spermatids and spermatozoa, concomitantly with AZ3, suggests that both proteins may play an important role in regulating polyamine levels during spermiogenesis