Fluorometric assay for alcohol sulfotransferase.
A sensitive fluorometric assay was developed for alcohol sulfotransferase (AST). This was the first continuous fluorometric assay reported for AST. It used 3'-phosphoadenosine 5'-phosphosulfate regenerated from 3-phosphoadenosine 5'-phosphate by a recombinant phenol sulfotransferase (PST) using 4-methylumbelliferyl sulfate as the sulfuryl group donor. The recombinant PST did not use the alcohol substrate under the designed condition, and the sensitivity for AST activity was found to be comparable to that of radioactive assay as reported in the literature. The change of fluorescence intensity of 4-methylumbelliferone corresponded directly to the amount of active AST and was sensitive enough to measure nanogram or picomole amounts of the enzyme activity. This fluorometric assay was used to determine the activities of AST as purified form and in crude extracts of pig liver, rat liver, and Escherichia coli. Some properties of human dehydroepiandrosterone sulfotransferase were determined by this method and were found to be comparable to published data. Under similar assay conditions, the contaminated activities of arylsulfatase in crude extracts were also determined. This method not only is useful for the routine and detailed kinetic study of this important class of enzymes but also has the potential for the development of a high-throughput procedure using microplate reader.
Chen
Wei-Ti
WT
Institute of Biological Science and Technology, College of Biological Science and Technology, National Chiao Tung University, Hsinchu, Taiwan, ROC.
Liu
Ming-Chih
MC
Yang
Yuh-Shyong
YS
D000244:Adenosine Diphosphate / Q000378:metabolism*; D000818:Animals; D004926:Escherichia coli / Q000201:enzymology; D006801:Humans; D006923:Hymecromone / Q000031:analogs & derivatives* / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D010724:Phosphoadenosine Phosphosulfate / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015238:Sulfotransferases / Q000378:metabolism*; D013462:Sulfur Radioisotopes; D013552:Swine
D011994:Recombinant Proteins; D013462:Sulfur Radioisotopes; C019492:4-methylumbelliferyl sulfate; D006923:Hymecromone; D010724:Phosphoadenosine Phosphosulfate; D000244:Adenosine Diphosphate; C010840:adenosine 3'-phosphate-5'-phosphate; D015238:Sulfotransferases; C028152:alcohol sulfotransferase