Fluorescence assay for protein post-translational tyrosine sulfation

Bo Han Chen, Chen Chu Wang, Lu Yi Lu, Kuo Sheng Hung, Yuh Shyong Yang

研究成果: 雜誌貢獻文章同行評審

9 引文 斯高帕斯(Scopus)

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.

原文英語
頁(從 - 到)1425-1429
頁數5
期刊Analytical and Bioanalytical Chemistry
405
發行號4
DOIs
出版狀態已發佈 - 2013

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry

指紋 深入研究「Fluorescence assay for protein post-translational tyrosine sulfation」主題。共同形成了獨特的指紋。

引用此