On the understanding of the interactions between ornithine decarboxylase (ODC) and the regulatory protein antizyme (AZ) in the feedback control of polyamine metabolism at the molecular level

• Main Author: Choe, Chungyoul
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01-01-1997
• Subjects: Molecular biology
• ISBN: 0591431947; 9780591431940

The polyamines putrescine, spermidine and spermine are essential compounds for cellular life, and their concentrations are strictly controlled by enzymes, polyamine transport and feedback mechanisms. This study was aimed at increasing our understanding of omithine decarboxylase (ODC), the rate-limiting enzyme in the polyamine biosynthetic pathway in cells, and AZ protein, the regulatory protein in polyamine feedback control. DH23b cells, selected from HTC parental cells by their resistance to the ODC inhibitor DFMO, exhibit amplification of the ODC gene which results in an increased biosynthesis of stable ODC protein. However, the AZ gene was unchanged in terms of its copy number and banding pattern, while multiple bands of AZ protein were rapidly induced in response to putrescine. In an initial study to understand the structural relationship between these multiple bands of AZ, their purification was attempted. ODC is degraded by the 26S proteasome in an AZ-dependent manner, and the C-terminus of ODC is important for its degradation. This study was carried out to determine what structural feature of the C-terminus renders ODC unstable. Rat ODC exhibited rapid degradation in rabbit reticulocyte lysates while remaining stable in the wheat germ system. HMOA and DH23b have stable forms of ODC which have Trp at amino acid 441 and exhibit enhanced electrophoretic mobility in SDS-PAGE. The latter characteristic suggests the possibility of a C-terminal truncation in these cell lines. However, amino acid sequencing of the C-terminus indicates that post-translational truncation does not occur in 441 Trp ODC. Further, the efficiency by which AZ exposes the C-terminus of ODC was tested. According to the releasing rate of the C-terminus amino acids, AZ does not effect exposure of the C-terminus. AZ induced by polyamines was studied extensively as a regulatory factor for post-translational ODC degradation and for polyamine transport. In addition, there is a possibility that AZ inhibits ODC translation or is involved in cotranslational degradation. According to this study, AZ induces translational degradation of stable and unstable ODC rather than inhibiting translation. Further, mutant AZ, in which the destabilization domain required for posttranslational degradation of ODC is deleted, decreased cotranslational degradation.