Yeast ornithine decarboxylase and antizyme form a 1:1 complex in vitro: purification and characterization of the inhibitory complex.

ABSTRACT

Saccharomyces cerevisiae antizyme (AZ) resembles mammalian AZ in its mode of synthesis by translational frameshifting and its ability to inhibit and facilitate the degradation of ornithine decarboxylase (ODC). Despite many studies on the interaction of AZ and ODC, the ODCAZ complex has not been purified from any source and thus clear information about the stoichiometry of the complex is still lacking. In this study we have studied the yeast antizyme protein and the ODCAZ complex. The far UV CD spectrum of the full-length antizyme shows that the yeast protein consists of 51% ß-sheet, 19% α-helix, and 24% coils. Surface plasmon resonance analyses show that the association constant (K(A)) between yeast AZ and yeast ODC is 6×10(7) (M(-1)). Using purified His-tagged AZ as a binding partner, we have purified the ODCAZ inhibitory complex. The isolated complex has no ODC activity. The molecular weight of the complex is 90 kDa, which indicates a one to one stoichiometric binding of AZ and ODC in vitro. Comparison of the circular dichroism (CD) spectra of the two individual proteins and of the ODCAZ complex shows a change in the secondary structure in the complex.