A deadenylase assay by size-exclusion chromatography.
A deadenylase assay by size-exclusion chromatography.
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尺寸排阻色谱法测定脱腺苷酶
DOI：
10.1371/journal.pone.0033700
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Yan YB
中科院分区：
文献类型：
--
作者：
He GJ;Yan YB
3′端poly（A）尾的缩短，也称为去腺苷酸化，对mRNA加工、运输、翻译和降解的调节至关重要。去腺苷酸化过程是由去腺苷酸酶完成的，去腺苷酸酶特异性地催化真核生物mRNA 3′-末端的poly（A）尾的去除并释放作为产物的5′-AMP。为了实现它们的生理功能，所有去腺苷化酶都具有许多结合伴侣，这些结合伴侣可以调节它们的催化性质或将它们募集到各种蛋白质复合物中。为了研究不同伴侣的作用，重要的是开发新的可以在体内或体外应用的去腺苷酶测定法。在本研究中，我们建立了尺寸排阻色谱法（SEC）去腺苷酶测定法。SEC分析表明，poly（A）或oligo（A）底物和产物AMP可以成功地分离和定量。通过定量腺苷酸的生成量，可以得到腺苷酸酶的酶学参数。当使用商业聚（A）作为底物时，观察到双相催化过程，这可能与商业样品中聚（A）的两种不同状态相关。不同批次的市售聚（A）具有不同的粒径分布，对去腺苷酸酶降解的反应也不同。也可以使用SEC测定法通过监测底物的状态以及AMP和A2的生成动力学来研究进行性或分布性的去腺苷化模式。SEC测定适用于使用纯化的酶的简单样品和复杂的酶反应条件，例如使用蛋白质混合物或粗细胞提取物作为样品。通过构建不同的SEC图谱，可以成功地消除在254 nm处有吸收的溶质的影响。
The shortening of the 3′-end poly(A) tail, also called deadenylation, is crucial to the regulation of mRNA processing, transportation, translation and degradation. The deadenylation process is achieved by deadenylases, which specifically catalyze the removal of the poly(A) tail at the 3′-end of eukaryotic mRNAs and release 5′-AMP as the product. To achieve their physiological functions, all deadenylases have numerous binding partners that may regulate their catalytic properties or recruit them into various protein complexes. To study the effects of various partners, it is important to develop new deadenylase assay that can be applied either in vivo or in vitro. In this research, we developed the deadenylase assay by the size-exclusion chromatography (SEC) method. The SEC analysis indicated that the poly(A) or oligo(A) substrate and the product AMP could be successfully separated and quantified. The enzymatic parameters of deadenylase could be obtained by quantifying the AMP generation. When using the commercial poly(A) as the substrate, a biphasic catalytic process was observed, which might correlate to the two distinct states of poly(A) in the commercial samples. Different lots of commercial poly(A) had dissimilar size distributions and were dissimilar in response to the degradation of deadenylase. The deadenylation pattern, processive or distributive, could also be investigated using the SEC assay by monitoring the status of the substrate and the generation kinetics of AMP and A2. The SEC assay was applicable to both simple samples using the purified enzyme and complex enzyme reaction conditions such as using protein mixtures or crude cell extracts as samples. The influence of solutes with absorption at 254 nm could be successfully eliminated by constructing the different SEC profiles.
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