Drazic Adrian, Arnesen Thomas
Department of Molecular Biology, University of Bergen, Thormøhlensgate 55, N-5020, Bergen, Norway.
Department of Surgery, Haukeland University Hospital, N-5021, Bergen, Norway.
Methods Mol Biol. 2017;1574:1-8. doi: 10.1007/978-1-4939-6850-3_1.
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]-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.
N 端乙酰化是最丰富的共翻译和翻译后蛋白质修饰之一，从原核生物到真核生物都保守存在。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。我们在此描述一种基于同位素标记的[C] - 乙酰辅酶 A 的乙酰化测定方法，可用于在体外测定 N - 乙酰转移酶（NATs）的微弱催化活性。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共免疫沉淀酶，还能测定各种细胞裂解物的体外活性。尽管被标记为一种老式的生化方法，但它是寻找新的潜在 N 端乙酰转移酶候选物的催化活性和确定肽特异性的理想方法。
