Ornithine decarboxylase

Expression and regulation in rat brain and in transgenic mice

Pekka Kilpeläinen

Department of Biochemistry, University of Oulu

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.

Table of Contents
Acknowledgements
Abbreviations
List of original articles
1. Introduction
2. Review of the literature
2.1. Polyamines
2.1.1. Structure and properties
2.1.2. Physiological roles
2.1.3. Polyamine metabolism in mammals
2.1.4. Polyamines in the central nervous system
2.1.5. Polyamines and the regulation of inward rectifying potassium channels
2.1.6. Polyamines and cancer
2.2. Ornithine decarboxylase
2.2.1. Enzyme protein and catalytic activity
2.2.2. Gene and mRNA
2.2.3. Regulation
2.3. Antizyme
2.3.1. Protein and functions
2.3.2. Gene family
2.3.3. Regulation
3. Aims of the present study
4. Materials and Methods
4.1. Assay of ornithine decarboxylase and antizyme activities (I, II, IV)
4.2. Preparation of brain extracts (I)
4.3. Construction of plasmids and site directed mutagenesis (II)
4.4. Cell culture and DNA transfection (II)
4.5. Northern blot analysis (II)
4.6. Generation and analysis of transgenic mice (IV)
4.7. In situ hybridization (III)
4.8. Immunocytochemistry (III)
4.9. Other methods
5. Results
5.1. Detection and characterization of GTP-activatable ornithine decarboxylase in the rat brain (I)
5.2. Expression of ornithine decarboxylase and antizyme in the rat brain (III)
5.3. Site-directed mutagenesis and analysis of D233V-mutated ornithine decarboxylase (II)
5.4. Generation and analysis of ornithine decarboxylase expressing transgenic mice (IV)
6. Discussion
6.1. GTP-activatable ornithine decarboxylase in the rat brain
6.2. Expression of ornithine decarboxylase and antizyme in the rat brain
6.3. Mutation of aspartate-233 to valine in mouse ornithine decarboxylase
6.4. Ornithine decarboxylase expressing transgenic mice
6.5. Conclusions
References
List of Tables
1. Semiquantitative evaluation of ODC and antizyme mRNA distribution in rat brain.
List of Figures
1. Biosynthesis and interconversion of polyamines. The enzymes catalyzing reactions are: 1. ATP:L-methionine S-adenosyltransferase (EC 2.5.1.6), 2. S-adenosylmethionine decarboxylase (EC 4.1.1.50), 3. Ornithine decarboxylase (EC 4.1.1.17), 4. Spermidine synthase (EC 2.5.1.16), 5. Spermine synthase (EC 2.5.1.22), 6. Spermidine/spermine N1-acetyltransferase (EC 2.3.1.57), and 7. Polyamine oxidase (EC 1.4.3.4)
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  Acknowledgements

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