The polyamines, putrescine, spermidine, and spermine, are major polybasic compounds in all living cells. These amines are important for many systems related to growth and differentiation. For many years we have been studying how these polyamines are synthesized, how their biosynthesis and degradation are regulated, their physiologic functions, how they act in vivo, and the structure of the various biosynthetic enzymes. For this purpose we have constructed null mutants in each of the biosynthetic steps in both Escherichia coli and Saccharomyces cerevisiae, and have prepared overexpression systems for the biosynthetic enzymes. Our overall studies have aimed at the use of these mutants to elucidate the physiological functions of the polyamines.? ? Our current studies are concerned with the interaction of yeast antizyme and yeast ornithine decarboxylase. This is a most unusual regulatory sustem. Antizyme ineracts with ornithine decarboxylase, resulting in inhibition of its activity. Antizyme also acts to target the ornithine decarboxylase protein to the proteasome for degradation; this degradation is unusual in not involving ubiquitination. Biosynthesis of antizyme is most unusual in requiring a frameshift in translation, in order to bypass a stop codon; this frameshift only occurs in the presence of increased spermdine. This unusual requirement for a frameshift has been conserved throughout evolution, even though there is considerable variation in the protein sequences in different organisms.? ? In our current studies we have constructed a 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 he two proteins. Preliminary studies have already been carried out with light scattering techniquesto find out the bioophysical properties of these two enzymes separately as a background for subsequent studies on their interactions. Preliminary characterization with CD spectra were also performed in order to study the secondary structure of these two proteins both individually and when resent as a complex. We have also continued attempts to crystallize the two proteins, especially since there is no x-ray structure available for the full lenghth yeast antizyme protein.
