2nd project summary: [unreadable] [unreadable] This project aims to investigate the unique regulation of ornithine decarboxylase (ODC), the key rate limiting enzyme of polyamine biosynthesis. The regulation of ODC in yeast and other eukaryotes is rather unusual involving a unique protein termed antizyme. This unique regulation involves polyamine induced synthesis of antizyme by +1 translational frameshifting and binding of the antizyme to ornithine decarboxylase.[unreadable] [unreadable] In our current studies we have constructed plasmids for both ornithine decarboxylase and for antizyme (with the in-frame sequence), and 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 purified proteins were also used to find out the binding affinity using biochemical assays. Our in vitro data demonstrated that the yeast ODC is normally present as an active homodimer, yeast antizyme binds to ODC and dissociates the homodimer and forms a heterodimer containing one molecule each of ODC and antizyme. This heterodimer has no activity of its own and can be dissociated by high ionic strength. The molecular weight of each species was determined by gel filtration and light scattering. The ODC homodimer is 104 kDa, antizyme 38 kDa (present as monomer) and the ODC:antizyme heterodimer 90 kDa. We have also performed the CD spectra of each protein (complex and individual) and the data suggest no change in secondary structure due to antizyme binding to ODC, although antizyme binding changes the tertiary structure of the protein by dissociating the dimer. The purified antizyme-ODC complex is being used for crystallization studies.