標題: Fluorescence assay for protein post-translational tyrosine sulfation
作者: Chen, Bo-Han
Wang, Chen-Chu
Lu, Lu-Yi
Hung, Kuo-Sheng
Yang, Yuh-Shyong
生物科技學系
Department of Biological Science and Technology
關鍵字: Protein sulfation;Tyrosylprotein sulfotransferase (TPST);Phenol sulfotransferase (PST);Fluorescence enzyme assay
公開日期: 1-二月-2013
摘要: We developed a fluorescent assay to conveniently determine the kinetics of protein sulfation, which is essential for understanding interface between protein sulfation and protein-protein interactions. Tyrosylprotein sulfotransferase (TPST) catalyzes protein sulfation using 3'-phosphate 5'-phosphosulfate (PAPS) as sulfuryl group donor. In this report, PAPS was regenerated following sulfuryl group transfer between adenosine 3',5'-diphosphate and 4-methylumbelliferyl sulfate catalyzed by phenol sulfotransferase (PST). The TPST and PST coupled enzyme platform continuously generated fluorescent 4-methylumbelliferone (MU) that was used to real-time monitor protein sulfation. Using a recombinant N utilization substance protein A fused Drosophila melanogaster tyrosylprotein sulfotransferase, we demonstrated that the activity of TPST determined through MU fluorescence directly correlated with protein sulfation. Kinetic constants obtained with small P-selectin glycoprotein ligand-1 peptide (PSGL-1 peptide, MW 1541) or its large glutathione S-transferase fusion protein (GST-PSGL-1, MW 27833) exhibited significant variation. This assay can be further developed to a high-throughput method for the characterization of TPSTs and for the identification and screening of their protein substrates.
URI: http://dx.doi.org/10.1007/s00216-012-6540-3
http://hdl.handle.net/11536/21027
ISSN: 1618-2642
DOI: 10.1007/s00216-012-6540-3
期刊: ANALYTICAL AND BIOANALYTICAL CHEMISTRY
Volume: 405
Issue: 4
起始頁: 1425
結束頁: 1429
顯示於類別:期刊論文


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  1. 000313735000028.pdf