Yeast ornithine decarboxylase and antizyme form a 1:1 complex in vitro: purification and characterization of the inhibitory complex.
Yeast ornithine decarboxylase and antizyme form a 1:1 complex in vitro: purification and characterization of the inhibitory complex.
复制标题
酵母鸟氨酸脱羧酶和抗酶在体外形成 1:1 复合物：抑制复合物的纯化和表征。
DOI：
10.1016/j.bbrc.2011.01.113
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发表时间：
2011
影响因子：
3.1
通讯作者：
Masison,DanielC
中科院分区：
文献类型：
--
作者：
Chattopadhyay,ManasK;Fernandez,Cristina;Sharma,Deepak;McPhie,Peter;Masison,DanielC
酿酒酵母（Saccharomyces cerevisiae， AZ）通过翻译移框合成的方式与哺乳动物的AZ相似，并且具有抑制和促进鸟氨酸脱羧酶（ODC）降解的能力。尽管对AZ和ODC的相互作用进行了许多研究，但ODC:AZ配合物尚未从任何来源纯化，因此仍然缺乏有关该配合物化学计量学的明确信息。在本研究中，我们研究了酵母抗酶蛋白和ODC:AZ复合物。全长抗酶的远紫外CD光谱表明，酵母蛋白由51%的β-片，19%的α-螺旋和24%的线圈组成。表面等离子体共振分析表明，酵母AZ和ODC之间的关联常数（KA）为6×107（M−1）。使用纯化的his标记的AZ作为结合伙伴，我们纯化了ODC:AZ抑制复合物。分离的配合物没有ODC活性。该配合物的分子量为90kDa，表明AZ和ODC在体外具有一对一的化学计量结合。比较两种单独蛋白和ODC:AZ配合物的圆二色性（CD）光谱，发现该配合物的二级结构发生了变化。
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 (KA) between yeast AZ and yeast ODC is 6×107(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 90kDa, 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.