Kumar Akhilesh, Zhang Fangliang
Department of Botany, Banaras Hindu University, Varanasi, UP, India.
Department of Molecular & Cellular Pharmacology, University of Miami Leonard M. Miller School of Medicine, Miami, FL, USA.
Methods Mol Biol. 2023;2620:71-80. doi: 10.1007/978-1-0716-2942-0_9.
Here, we describe an antibody-based method to evaluate the enzymatic activity of arginyltransferase1 (Ate1). The assay is based on the arginylation of a reporter protein, which contains the N-terminal peptide of beta-actin, a known endogenous substrate of Ate1, and a C-terminal GFP. The arginylation level of the reporter protein is determined on an immunoblot with an antibody specific for the arginylated N-terminus, while the total amount of substrate is evaluated with anti-GFP antibody. This method can be used to conveniently and accurately examine the Ate1 activity in yeast and mammalian cell lysates. Moreover, the effect of mutation on Ate1 critical residues and effect of stress and other factors on Ate1 activity can also be successfully determined with this method.
在此，我们描述了一种基于抗体的方法来评估精氨酰转移酶1（Ate1）的酶活性。该测定基于一种报告蛋白的精氨酰化，该报告蛋白包含β-肌动蛋白的N端肽（Ate1的一种已知内源性底物）和C端绿色荧光蛋白（GFP）。使用针对精氨酰化N端的特异性抗体通过免疫印迹法测定报告蛋白的精氨酰化水平，同时用抗GFP抗体评估底物的总量。该方法可用于方便且准确地检测酵母和哺乳动物细胞裂解物中的Ate1活性。此外，用该方法还能成功确定Ate1关键残基突变的影响以及应激和其他因素对Ate1活性的影响。
