title: Golgi-resident PAP-specific 3'-phosphatase-coupled sulfotransferase assays.
abstract: Sulfotransferases are a large group of enzymes that transfer a sulfonate group from the donor substrate, 3'-phosphoadenosine-5'-phosphosulfate (PAPS)(1), to various acceptor substrates, generating 3'-phosphoadenosine-5'-phosphate (PAP) as a by-product. A universal phosphatase-coupled sulfotransferase assay is described here. In this method, Golgi-resident PAP-specific 3'-phosphatase (gPAPP) is used to couple to a sulfotransferase reaction by releasing the 3'-phosphate from PAP. The released phosphate is then detected using malachite green reagents. The enzyme kinetics of gPAPP have been determined, which allows calculation of the coupling rate, the ratio of product-to-signal conversion, of the coupled reaction. This assay is convenient, as it eliminates the need for radioisotope labeling and substrate-product separation, and is more accurate through removal of product inhibition and correction of the results with the coupling rate. This assay is also highly reproducible, as a linear correlation factor above 0.98 is routinely achievable. Using this method, we measured the Michaelis-Menten constants for recombinant human CHST10 and SULT1C4 with the substrates phenolphthalein glucuronic acid and α-naphthol, respectively. The activities obtained with the method were also validated by performing simultaneous radioisotope assays. Finally, the removal of PAP product inhibition by gPAPP was clearly demonstrated in radioisotope assays.
authors.affiliation: R&D Systems, Inc., Minneapolis, MN 55413, United States.
authors.forename: Brittany
authors.initials: B
authors.lastname: Prather
authors.forename: Cheryl M
authors.initials: CM
authors.lastname: Ethen
authors.forename: Miranda
authors.initials: M
authors.lastname: Machacek
authors.forename: Zhengliang L
authors.initials: ZL
authors.lastname: Wu
mesh_terms: D057075:Enzyme Assays; D006056:Golgi Apparatus / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012394:Rosaniline Dyes / Q000737:chemistry; D013379:Substrate Specificity; D015238:Sulfotransferases / Q000235:genetics / Q000378:metabolism*; D000097811:Carbohydrate Sulfotransferases
chemical_list: D010744:Phosphoric Monoester Hydrolases; D011994:Recombinant Proteins; D012394:Rosaniline Dyes; D015238:Sulfotransferases; C005095:malachite green; C022321:PAPS sulfotransferase; C499549:CHST10 protein, human; D000097811:Carbohydrate Sulfotransferases