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.

Fig. 1. Purification of yeast antizyme protein

(A) SDS-PAGE showing the purification of his-tagged antizyme. The yeast AZ protein was overexpressed in E. coli and purified using cobalt column (TALON) as mentioned in Materials and Methods. Aliquots of samples from different steps were run on SDS-PAGE together with protein standard. (B) The native molecular weight of his-tagged antizyme protein was determined by size exclusion chromatography followed by light scattering. Purified antizyme protein (250 µl) from cobalt column at 1mg/ml concentration was loaded into a superdex-200 gel filtration column (GL 30/200) equilibrated with phosphate base saline and was run at a flow rate of 0.5 ml/min. The top panel shows the light scattering at 90° angles and the bottom panel shows optical density at 280 nm. The average molecular weight was determined by the measurement of scattered light in comparison to the protein concentration. The system was calibrated using bovine serum albumin and ovalbumin as standards.

Fig. 2. Inhibition of ornithine decarboxylaseby yeast antizyme and affinity constants of protein-protein interaction

(A) 2 pmole of ODC was incubated in the presence and absence of antizyme, and the ODC activity remaining after incubation was determined by ¹⁴CO₂ trapped in Ba(OH)₂ soaked filter paper. Assays were performed in triplicates and the average data are presented here. (B) Sensogram showing the binding of different concentration of ODC protein (0.125, 0.25 and 0.5 µM) to the surface immobilized antizyme protein, as obtained from surface plasmon resonance analyses in a Biacore 2000 as described in Materials and Methods.

Fig. 3. Stoichiometry of yeast ODC:AZ complex

(A) Purified ODC was incubated overnight with His-tagged purified antizyme in different molar ratios as indicated in the figure. The resulting complex was trapped on cobalt (TALON) column and purified as described in Materials and Methods. An aliquot of the purified sample was run on SDS-PAGE together with protein standard. (B). The purified ODC: AZ complex was assayed on SEC-lightscattering to determine the native molecular weight of the ODC: AZ complex. The top panel is showing the dynamic (multi angle) light scattering, the bottom panel is showing the refractive index for protein concentration determination as determined by a refractive index detector (Optilab Rex).

Fig. 4. CD spectra of purified AZ, ODC and ODC: AZ complex show a change in secondary structure in the complex

Far UV (260–190 nm) CD spectra of purified antizyme (A) and ornithine decarboxylase (B) proteins. Antizyme or ornithine decarboxylase proteins at 0.1 mg/ml were run on a CD (JASCO) and the mean ellipticity of the molecule was determined. The data was analyzed to calculate the percentage of α-helices and β-sheets present in antizyme protein as mentioned in the text. (C). Far UV (260–190 nm) CD spectra of purified ODC: AZ complex (0.1 mg/ml) is shown here. The data were calculated and compared with the sum of the CD spectra of 1:1 molar ratios two molecules.