Biophysical studies on the interaction of antizyme and ornithine decarboxylase

本项目旨在研究鸟氨酸脱羧酶（ODC）的独特调控，ODC是多胺生物合成的关键限速酶，涉及一种独特的蛋白质，称为抗酶。Antizyme与ODC结合，抑制ODC活性，并靶向26 S蛋白酶体降解ODC，而不进行多聚泛素化。在我们目前的研究中，我们一直在研究这种相互作用的化学计量和表征。为了这个目的，我们已经纯化了酵母鸟氨酸脱羧酶和酵母抗酶的同质性都试图结晶和生物物理研究的两种蛋白质的相互作用。最近，我们已经开发了纯化ODC：抗酶复合物的方法，并通过各种生物物理和生物化学技术（凝胶过滤，尺寸排阻-静态光散射，动态光散射，平衡超离心，CD光谱）研究了它们的相互作用。酵母AZ与酵母ODC的结合亲和力为5 × 10 ~（-11）M。使用纯化的His标记的AZ作为结合伴侣，我们纯化了AZ：ODC异源二聚体，其没有ODC活性。复合物的天然分子量为90 kDa，这表明AZ和ODC在体外以1：1的化学计量结合。两个单独的蛋白质和ODC：AZ复合物的圆二色性（CD）光谱显示ODC或AZ的二级结构在相互结合后没有显着变化。这些结果表明，酵母抗酶和鸟氨酸脱羧酶形成异源二聚体，由每个单体组成，并且AZ：ODC复合物中的二级结构没有变化。

This project aims to investigate the unique regulation of ornithine decarboxylase (ODC), the key rate limiting enzyme of polyamine biosynthesis, involving a unique protein termed antizyme. Antizyme binds to ODC, inhibits ODC activity and targets the degradation of ODC by the 26S proteasome without poly-ubiquitination. In our current studies we have been studying the stoichiometry and characterization of this interaction. For this purpose we 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 binding affinity for yeast AZ to yeast ODC is 5X10-11 M. Using purified His-tagged AZ as a binding partner, we have purified the AZ:ODC heterodimer, which has no ODC activity. The native molecular weight of the complex is 90 kDa, which suggests a 1:1 stoichiometric binding of AZ and ODC in vitro. Circular dichroism (CD) spectra of the two individual proteins and the ODC:AZ complex show no significant change in the secondary structure of either ODC or AZ after binding to each other. These results indicate that yeast antizyme and ornithine decarboxylase form a heterodimer, consisting of each monomer and that there is no change in secondary structure in the AZ:ODC complex.