[14C]-Acetyl-Coenzyme A-Based In Vitro N-Terminal Acetylation Assay
[14C]-Acetyl-Coenzyme A-Based In Vitro N-Terminal Acetylation Assay
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DOI：
10.1007/978-1-4939-6850-3_1
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发表时间：
2017-01-01
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影响因子：
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通讯作者：
Arnesen, Thomas
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文献类型：
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作者：
Drazic, Adrian;Arnesen, Thomas
N 末端乙酰化是最丰富的蛋白质共翻译和翻译后修饰之一，从原核生物到真核生物都是保守的。这种修饰的功能后果是多方面的，从蛋白质折叠、稳定性、相互作用到亚细胞定位。我们在此描述了一种基于同位素标记的 [C-14]-乙酰基-辅酶 A 的乙酰化测定，可以在体外测定 NAT 的弱催化活性。它允许使用来自大肠杆菌的纯化重组酶或来自各种生物体的免疫共沉淀酶，以及测定各种细胞裂解物的体外活性。尽管被标记为老式生化方法，但它是寻找催化活性和定义新的潜在 N 末端乙酰转移酶候选物的肽特异性的理想方法。
N-terminal acetylation is one of the most abundant co-and posttranslational protein modifications, conserved from prokaryotes to eukaryotes. The functional consequences of this modification are manifold, ranging from protein folding, stability, and interaction to subcellular localization. We describe here an isotope-labeled [C-14]-acetyl-Coenzyme A-based acetylation assay, allowing the determination of weak catalytic activities of NATs in vitro. It allows the use of purified recombinant enzymes from Escherichia coli, or co-immunoprecipitated enzymes from various organisms, as well as the determination of the in vitro activity of various cell lysates. Although marked as an old-fashioned biochemical approach, it is the ideal method to hunt for catalytic activities and defining peptide specificities of new potential N-terminal acetyltransferase candidates.