This project aims to investigate the unique regulation of ornithine decarboxylase (ODC), the key rate limiting enzyme of polyamine biosynthesis, involving a unique protein termed antizyme. Antizyme binds to ODC, inhibits ODC activity and targets the degradation of ODC by the 26S proteasome without poly-ubiquitination. In our current studies we have been studying the stoichiometry and characterization of this interaction. For this purpose we have purified both yeast ornithine decarboxylase and yeast antizyme to homogeneity both for attempts at crystallization and for biophysical studies on the interactions of the two proteins. Recently we have developed methods to purify the ODC:antizyme complex and studied their interactions by various biophysical and biochemical techniques (Gel filtration, size exclusion-static-light scattering, dynamic light scattering, equilibrium ultracentrifugation, CD spectra). The binding affinity for yeast AZ to yeast ODC is 5X10-11 M. Using purified His-tagged AZ as a binding partner, we have purified the AZ:ODC heterodimer, which has no ODC activity. The native molecular weight of the complex is 90 kDa, which suggests a 1:1 stoichiometric binding of AZ and ODC in vitro. Circular dichroism (CD) spectra of the two individual proteins and the ODC:AZ complex show no significant change in the secondary structure of either ODC or AZ after binding to each other. These results indicate that yeast antizyme and ornithine decarboxylase form a heterodimer, consisting of each monomer and that there is no change in secondary structure in the AZ:ODC complex.
| Chattopadhyay, Manas K; Fernandez, Cristina; Sharma, Deepak et al. (2011) Yeast ornithine decarboxylase and antizyme form a 1:1 complex in vitro: purification and characterization of the inhibitory complex. Biochem Biophys Res Commun 406:177-82 |
