Human 3′-phosphoadenosine 5′-phosphosulfate synthetase: Radiochemical enzymatic assay, biochemical properties, and hepatic variation

Zhen Hua Xu, Thomas C. Wood, Araba A. Adjei, Richard M Weinshilboum

Research output: Contribution to journalArticle

16 Citations (Scopus)

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 languageEnglish (US)
Pages (from-to)172-178
Number of pages7
JournalDrug Metabolism and Disposition
Volume29
Issue number2
StatePublished - 2001

Fingerprint

Enzyme Assays
Ligases
Assays
Phosphoadenosine Phosphosulfate
Liver
Adenosine Triphosphate
Sulfates
Estradiol
Cytosol
Enzymes
Sulfotransferases
Biopsy
Enzyme activity
Xenobiotics
Substrates
PAPS synthetase
Genetic Association Studies
Biotransformation
Brain
Cerebral Cortex

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 journalArticle

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