title: 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 ODC:AZ 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 ODC:AZ complex. The far UV CD spectrum of the full-length antizyme shows that the yeast protein consists of 51% β-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 ODC:AZ 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 ODC:AZ complex shows a change in the secondary structure in the complex.
authors.affiliation: Laboratory of Biochemistry and Genetics, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, 8 Center Drive, Bldg. 8, Bethesda, MD 20892, USA. manasc@intra.niddk.nih.gov
authors.forename: Manas K
authors.initials: MK
authors.lastname: Chattopadhyay
authors.forename: Cristina
authors.initials: C
authors.lastname: Fernandez
authors.forename: Deepak
authors.initials: D
authors.lastname: Sharma
authors.forename: Peter
authors.initials: P
authors.lastname: McPhie
authors.forename: Daniel C
authors.initials: DC
authors.lastname: Masison
mesh_terms: D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics; D009955:Ornithine Decarboxylase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D065108:Ornithine Decarboxylase Inhibitors; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
chemical_list: C497099:Oaz1 protein, S cerevisiae; D065108:Ornithine Decarboxylase Inhibitors; D011506:Proteins; D029701:Saccharomyces cerevisiae Proteins; C031017:ornithine decarboxylase antizyme; D009955:Ornithine Decarboxylase