High-Throughput Arginylation Assay in Microplate Format.
Here we describe the biochemical assay for ATE1-mediated arginylation in microplate format, which can be applied to high-throughput screens for identification of small-molecule inhibitors and activators of ATE1, high-volume analysis of ATE1 substrates, and other similar applications. Originally, we have applied this screen to a library of 3280 compounds and identified two compounds which specifically affect ATE1-regulated processes in vitro and in vivo. The assay is based on in vitro ATE1-mediated arginylation of beta-actin's N-terminal peptide, but it can also be applied using other ATE1 substrates.
Department of Molecular Biology and Biotechnology, Tezpur University, Napaam, Sonitpur, Assam, India.
Sougata
S
Saha
Junling
J
Wang
Anna S
AS
Kashina
D019881:Aminoacyltransferases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D001120:Arginine / Q000378:metabolism*; D055808:Drug Discovery / Q000379:methods; D057166:High-Throughput Screening Assays / Q000379:methods; D011499:Protein Processing, Post-Translational; D013379:Substrate Specificity
D001120:Arginine; D019881:Aminoacyltransferases