Versatile O-GlcNAc Transferase Assay for High-Throughput Identification of Enzyme Variants, Substrates, and Inhibitors
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- 作者：Eun J. Kim ; Lara K. Abramowitz ; Michelle R. Bond ; Dona C. Love ; Dong W. Kang ; Hans F. Leucke ; Dae W. Kang ; Jong-Seog Ahn ; John A. Hanover
- 刊名：Bioconjugate Chemistry
- 出版年：2014
- 出版时间：June 18, 2014
- 年：2014
- 卷：25
- 期：6
- 页码：1025-1030
- 全文大小：356K
- 年卷期：v.25,no.6(June 18, 2014)
- ISSN：1520-4812
文摘
The dynamic glycosylation of serine/threonine residues on nucleocytoplasmic proteins with a single N-acetylglucosamine (O-GlcNAcylation) is critical for many important cellular processes. Cellular O-GlcNAc levels are highly regulated by two enzymes: O-GlcNAc transferase (OGT) is responsible for GlcNAc addition and O-GlcNAcase (OGA) is responsible for removal of the sugar. The lack of a rapid and simple method for monitoring OGT activity has impeded the efficient discovery of potent OGT inhibitors. In this study we describe a novel, single-well OGT enzyme assay that utilizes 6 脳 His-tagged substrates, a chemoselective chemical reaction, and unpurified OGT. The high-throughput Ni-NTA Plate OGT Assay will facilitate discovery of potent OGT-specific inhibitors on versatile substrates and the characterization of new enzyme variants.