title: Fluorescence assay for protein post-translational tyrosine sulfation.
abstract: 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.
authors.affiliation: Department of Biological Science and Technology, National Chiao Tung University, Hsinchu, Taiwan.
authors.forename: Bo-Han
authors.initials: BH
authors.lastname: Chen
authors.forename: Chen-Chu
authors.initials: CC
authors.lastname: Wang
authors.forename: Lu-Yi
authors.initials: LY
authors.lastname: Lu
authors.forename: Kuo-Sheng
authors.initials: KS
authors.lastname: Hung
authors.forename: Yuh-Shyong
authors.initials: YS
authors.lastname: Yang
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D000818:Animals; D015239:Arylsulfotransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D029721:Drosophila Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000737:chemistry / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D006923:Hymecromone / Q000031:analogs & derivatives / Q000378:metabolism; D007700:Kinetics; D008163:Luminescent Measurements / Q000379:methods*; D011499:Protein Processing, Post-Translational; D015238:Sulfotransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism*
chemical_list: D029721:Drosophila Proteins; D006923:Hymecromone; D014443:Tyrosine; D000244:Adenosine Diphosphate; C010840:adenosine 3'-phosphate-5'-phosphate; D015238:Sulfotransferases; D015239:Arylsulfotransferase; C039079:protein-tyrosine sulfotransferase