Abstract
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 17β-[3H]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-catalized step, and because of its low Km 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 Km values for ATP of 0.26 and 0.62 mM, respectively, and for SO4 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.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 172-178 |
| Number of pages | 7 |
| Journal | Drug Metabolism and Disposition |
| Volume | 29 |
| Issue number | 2 |
| State | Published - 2001 |
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ASJC Scopus subject areas
- Pharmacology
- Toxicology
Cite this
Human 3′-phosphoadenosine 5′-phosphosulfate synthetase : Radiochemical enzymatic assay, biochemical properties, and hepatic variation. / Xu, Zhen Hua; Wood, Thomas C.; Adjei, Araba A.; Weinshilboum, Richard M.
In: Drug Metabolism and Disposition, Vol. 29, No. 2, 2001, p. 172-178.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Human 3′-phosphoadenosine 5′-phosphosulfate synthetase
T2 - Radiochemical enzymatic assay, biochemical properties, and hepatic variation
AU - Xu, Zhen Hua
AU - Wood, Thomas C.
AU - Adjei, Araba A.
AU - Weinshilboum, Richard M
PY - 2001
Y1 - 2001
N2 - 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 17β-[3H]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-catalized step, and because of its low Km 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 Km values for ATP of 0.26 and 0.62 mM, respectively, and for SO4 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.
AB - 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 17β-[3H]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-catalized step, and because of its low Km 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 Km values for ATP of 0.26 and 0.62 mM, respectively, and for SO4 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.
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M3 - Article
C2 - 11159808
AN - SCOPUS:0034746543
VL - 29
SP - 172
EP - 178
JO - Drug Metabolism and Disposition
JF - Drug Metabolism and Disposition
SN - 0090-9556
IS - 2
ER -