Human 3'-phosphoadenosine 5'-phosphosulfate synthetase: radiochemical enzymatic assay, biochemical properties, and hepatic variation.
Sulfation is a major pathway in the biotransformation of many drugs and other xenobiotic compounds. The sulfotransferase (SULT) enzymes that catalyze these reactions use 3'-phosphoadenosine 5'-phosphosulfate (PAPS) as a sulfate donor cosubstrate. The synthesis of PAPS from inorganic sulfate and ATP is catalyzed by PAPS synthetase (PAPSS). We previously cloned the genes for human PAPSS1 and PAPSS2 as a step toward pharmacogenetic studies of these enzymes. We have now developed a sensitive PAPSS radiochemical enzymatic assay for use in genotype-phenotype correlation analyses. This coupled assay uses the sulfation of 17beta-[(3)H]estradiol catalyzed by recombinant human SULT1E1 to measure PAPS, which has been generated by PAPSS during the initial step of the assay. SULT1E1 proved to be ideal for this application both because of its relative resistance to inhibition by ATP, a substrate for the PAPSS-catalyzed step, and because of its low K(m) values for both PAPS (58 nM) and estradiol (29 nM). After optimal PAPSS assay conditions had been established, substrate kinetic studies were performed with cytosol preparations from human liver and cerebral cortex, two tissues with very different expression patterns for PAPSS1 and PAPSS2 mRNA. Brain and liver cytosol PAPSS activities had apparent K(m) values for ATP of 0.26 and 0.62 mM, respectively, and for SO(4)(2-) of 0.08 and 0.31 mM, respectively. PAPSS activity was then measured in 83 human liver biopsy samples to determine the nature and extent of individual variation in this enzyme activity. An 18-fold variation was observed. This sensitive new radiochemical assay can now be used in pharmacogenetic studies of PAPSS in humans.
Xu
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Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Medical Foundation, Rochester, MN 55905-0001, USA.
Wood
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TC
Adjei
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Weinshilboum
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D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D019556:COS Cells / Q000201:enzymology; D004958:Estradiol / Q000378:metabolism; D005260:Female; D006801:Humans; D007206:Individuality; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008297:Male; D009097:Multienzyme Complexes / Q000378:metabolism*; D011869:Radioligand Assay; D013430:Sulfate Adenylyltransferase / Q000378:metabolism*; D015238:Sulfotransferases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014316:Tritium
D007527:Isoenzymes; D009097:Multienzyme Complexes; D014316:Tritium; D004958:Estradiol; D000255:Adenosine Triphosphate; C022322:PAPS synthetase; D013430:Sulfate Adenylyltransferase; D015238:Sulfotransferases